Methods for identifying compounds for inhibiting of neoplastic lesions, and pharmaceutical compositions containing such compounds

ABSTRACT

This invention provides pharmaceutical compositions containing compounds for the treatment of neoplasia in mammals. The phosphodiesterase inhibitory activity of a compound is determined along with COX inhibitory activity. Growth inhibitory and apoptosis inducing effects on cultured tumor cells are also determined. Compounds that exhibit phosphodiesterase inhibiton, growth inhibition and apoptosis induction, but prefereably not substantial prostaglandin inhibitory activity, are desirable for the treatment of neoplasia.

This application is a continuation-in part of U.S. patent applicationSer. No. 09/046,739 filed Mar. 24, 1998 which was a continuation-in-partof Ser. No. 08/866,027 filed May 30, 1997 (now U.S. Pat. No. 5,858,694).

BACKGROUND OF THE INVENTION

This application claims priority under 35 U.S.C. §120 to U.S. PatentApp. Ser. Nos. 08/866,027 and 09/046,739, filed May 30, 1997 and Mar.24, 1998, respectively.

This invention relates to the use of one or more forms ofphosphodiesterase type 2 (“PDE2”) and phosphodiesterase type 5 (“PDE5”)and/or protein kinase G to identify compounds useful for the treatmentand prevention of pre-cancerous and cancerous lesions in mammals, and topharmaceutical compositions containing such compounds, as well as totherapeutic methods of treating neoplasia with such compounds.

Currently, non-surgical cancer treatment involves administering one ormore highly toxic chemotherapeutics or hormonal therapies to the patientafter her cancer has progressed to a point where the therapeuticbenefits of chemotherapy/hormonal outweigh its very serious sideeffects. Such side effects are well known to any oncologist, and varyfrom drug to drug. However, standard chemotherapeutics are typicallyused only for short periods of time, often alternating chemotherapy withperiods off treatment, so as not to overwhelm the patient with drug sideeffects. Thus, given the risk-benefit trade-off, side effects typicallypreclude starting chemotherapy when patients exhibit precancerouslesions, or continuing chemotherapy or hormonal therapy on a chronicbasis after frank cancer has been eliminated in an attempt to preventits re-occurrence.

Beginning a decade or so ago, a glimmer of hope began to appear from anunexpected source: non-steroidal anti-inflammatory drugs (“NSAIDs”).Cancer and precancer research is replete with publications that describevarious biochemical molecules that are over-expressed in neoplastictissue, leading one group after another to research whether specificover-expressed molecules are responsible for the disease, and whether,if such over-expression were inhibited, neoplasia could be alleviated.For example, in familial adenomatous polyposis (“FAP”), Waddell in 1983(Waddell, W. R. et al., “Sulindac for Polyposis of the Colon,” Journalof Surgical Oncology, 24:83-87, 1983) hypothesized that sinceprostaglandins were over-expressed in such polyps, non-steroidalanti-inflammatory drugs (“NSAIDs”) should alleviate the conditionbecause NSAIDs inhibited prostaglandin synthetase PGE₂) activity. Thus,he administered the nonsteroidal anti-inflammatory drug (“NSAID”)sulindac (an inhibitor of PGE₂) to several FAP patients. Waddelldiscovered that polyps regressed and did not recur upon such therapy.PGE₂ inhibition results from the inhibition of cyclooxygenase (COX) byNSAIDs. The success by Waddell with sulindac and the PGE₂/COXrelationship seemingly confirmed the role of two other biochemicaltargets —PGE₂ and COX—in carcinogenesis, and the subsequent literaturereinforced these views.

The glimmer of hope for patients suffering from neoplasia was thatsulindac certainly exhibited far fewer side effects than conventionalchemotherapeutics or hormonals, and opened up the possibility oftreating cancer at earlier stages of the disease, and for longer periodsof time as compared with conventional chemotherapeutics. However, such ahope had to be tempered with the open question of whether a compoundsuch as sulindac could be used to treat frank cancer, given that Waddellhad only administered sulindac to patients with a pre-cancerouscondition, FAP.

That hope was also tempered by NSAIDs own sets of side effects. Sulindacand other NSAIDs when chronically administered, aggravate the digestivetract where PGE₂ Plays a protective role. In addition, when takenchronically, they exhibit side effects involving the kidney andinterference with normal blood clotting. As Waddell unfortunatelyexperienced, some of his sulindac patients stopped taking drug becauseof side effects (see Waddell, W. R. et al., “Sulindac for Polyposis ofthe Colon,” The American Journal of surgery, 157: 175-79, 1989), mostlikely returning to additional surgical interventions to control polypformation. Thus, for neoplasia patients, such drugs are not a practicalchronic treatment, e.g., for FAP, sporadic polyps or menpost-prostatectomy with rising PSAs (a rising PSA in such men indicatesthe recurrence of disease, which may not yet present as a frank, visiblecancer). These side effects also limit NSAIDs'use for any otherneoplasia indication requiring long-term drug administration. Morerecently, some have suggested that the COX-2 specific NSAIDs such ascelecoxib be used. However, the renal and other side effects of suchcompounds are believed to limit the dosing and length of treatment withsuch compounds for long-term anti-neoplastic indications. In addition,recently published data indicate that very high doses are needed fordrugs like celecoxib to achieve a marginal effect on polyps in onlypre-defined regions of colorectum. Perhaps more significant to coloncancer treatment is that it has been reported that certain colonicneoplasias (e.g., HCT-116) do not express COX-2, and that suchinhibitors are ineffective against such neoplasias (see, Sheng, et al.,“Inhibition of Human Colon Cancer Cell Growth By Selective Inhibition ofCyclooxygenase-2,” J. Clin. Invest., 99(9):2254-9, 1997).

Recent discoveries have lead scientists away from the COX/PGE₂ targets,since those targets may not be the primary (or perhaps even secondarytargets) to treat neoplasia patients successfully on a chronic basis.Pamukcu et al., in U.S. Pat. No. 5,401,774, disclosed that sulfonylcompounds, that have been reported to be practically devoid of PGE2 andCOX inhibition (and therefore not NSAIDs or anti-inflammatory compounds)unexpectedly inhibited the growth of a variety of neoplastic cells,including colon polyp cells. These sulfonyl derivatives have proveneffective in rat models of colon carcinogenesis, and one variant (nowreferred to as exisulind) has proven effective in human clinical trialswith FAP patients, and even more remarkably has shown effect in a frankcancer: prostate cancer itself, in a controlled clinical study presentedbelow. Furthermore, very recent research has convincingly establishedthat COX I and/or COX II are not expressed substantially in allneoplasias, diminishing the hope that a COX I or COX II specificinhibitor would be broadly therapeutically useful in neoplasia treatment(see, Lim et al., “Sulindac Derivatives Inhibit Growth and InduceApoptosis in Human Prostate Cancer Cell Lines,” Biochem. Pharmacology,Vol. 58, pp. 1097-1107 (1999) in press).

Thus, like so many other proteins over-expressed in neoplasias, PGE₂/COXover-expression may not be a cause of some neoplasias, rather aconsequence of some of them. But the combination of such discoveries,however, has raised the question about how do compounds such asexisulind (that have a range of activity against both COX and non-COXexpressing neoplasias) act? What do such compounds do to neoplasticcells?.

Piazza, et al. (in U.S. patent application Nos. 08/866,027 and09/046,739) discovered that compounds (such as exisulind) inhibitedcyclic-specific GMP phosphodiesterase (e.g., PDE5), and that other suchcompounds could be screened using that enzyme, which could lead to thediscovery of still other compounds that could be developed andformulated into anti-neoplastic pharmaceutical compositions. Suchpharmaceutica cmpositions can be highly anti-neoplastic and can bepractically devoid of side effects associated with conventionalchemotherapeutics, or even the side effects of COX or PGE2 inhibition,if one wanted to avoid such side effects. In addition, anti-neoplasticcGMP-specific PDE-inhibiting compounds can induce apoptosis (a form ofprogrammed cell death or suicide) in neoplastic cells, but not in normalcells. Thus, such new compounds have become referred to as a new classof antineoplastics known as selective apoptotic anti-neoplastic drugs(“SAANDs”). Accordingly, SAANDs have challenged several matters ofconventional wisdom: (1) that anti-neoplastic compounds cannot beeffective without also killing normal cells; (2) that COX's areresponsible for neoplasia; and (3) that prevention of colonic neoplasiaby NSAIDs is likely mediated by the inhibition of one or both types ofCOX.

New research presented below has, however, shown that not all compoundsexhibiting classic PDE5 inhibition induce apoptosis in neoplastic cells.For example, the well-known PDE5 inhibitors, zaprinast and sildenafil,do not singly induce apoptosis, or even inhibit neoplastic cell growthin our hands. However, because pro-apoptotic PDE5 inhibitors inducedapoptosis selectively (i.e., in neoplastic but not in normal cells), andcould do so without substantial COX inhibition, the usefulness of PDE5as a screening tool for desirable anti-neoplastic compounds isunquestioned.

However, an enhancement to the PDE5 screening method to findanti-neoplastic, pro-apoptotic but safe compounds is desirable so thatnew pharmaceutical compositions can be formulated for therapeutic use inthe treatment of neoplasia, including pre-cancer and cancer.

SUMMARY OF THE INVENTION

In the course of researching why some PDE5 inhibitors singly inducedapoptosis while others did not, we uncovered a form of cyclicGMP-specific phosphodiesterase activity, not previously described. Thisnew phosphodiesterase activity was previously uncharacterized. . Withoutbeing limited to a specific theory, we believe this novel PDE activitymay be a novel conformation of PDE2 that substantially lackscAMP-hydrolyzing activity, i.e. it is cGMP-specific. Classic PDE2 is notcGMP-specific (it also hydrolyzes cAMP), classic PDE2 is also found inneoplastic cells. This new PDE and PDE2 are useful in screeningpharmaceutical compounds for desirable anti-neoplastic properties.Basically, eoplastic cells when PDE5 and the PDE2 activity (in its noveland conventional conformations) are inhibited by an anti-neoplasticPDE5-inhibiting compound, the result is apoptosis. When only PDE5 isinhibited (but not the several forms of PDE2), apoptosis does not occur.

In its broadest aspects, this new PDE conformation has activitycharacterized by:

(a) cGMP specificity over cAMP

(b) positive cooperative kinetic behavior in the presence of cGMPsubstrate;

(c) submicromolar affinity for cGMP; and

(d) insensitivity to incubation with purified cGMP-dependent proteinkinase

Other characteristics of this novel PDE include: it has reducedsensitivity to inhibition by zaprinast and E4021, it can be separatedfrom classical PDE5 activity by anion-exchange chromatography, it is notactivated by calcium/calmodulin, and it is insensitive to rolipram,vinpocetine and indolidan.

Another embodiment of this invention involves evaluating whether acompound causes an increase in cGMP-dependent protein kinase G (“PKG”)activity and/or a decrease of β-catenin in neoplastic cells. It has beenfound that unexpected characteristics of SAANDs include the elevation ofPKG activity and a decrease in β-catenin in neoplastic cells exposed toa SAAND. We believe that the elevation of PKG activity is due at leastin part by the increase in cGMP caused by SAANDs inhibition of theappropriate PDEs, as described above. The other characteristics ofSAANDs are (1) inhibition of PDE5 as reported in the '694 Patent above,(2) inhibition of the novel cGMP-specific PDE conformation, (3)inhibition of PDE2; (4) the fact that they increase intracellular cGMPin neoplastic cells, and (5) the fact that they decrease cAMP levels insome types of neoplastic cells.

Thus, one embodiment of the novel method of this invention is evaluatinghether a compound causes PKG activity to elevate in neoplastic cells andwhether hat compound inhibits PDE5. Another embodiment of the novelscreening method of his invention is evaluating whether a compound thatcauses PKG activity to elevate in neoplastic cells and whether thatcompound inhibits the novel cGMP-specific PDE described above and/orPDE2. Still a third embodiment is evaluating whether a compound causesPKG activity to elevate in neoplastic cells and whether that compoundcauses cGMP to rise in neoplastic cells and/or causes cAMP levels tofall. Compounds successfully evaluated in such fashions have applicationas SAANDs.

Among other things, this invention relates to novel in vitro and in vivomethods for selecting compounds for their ability to treat and preventneoplasia, especially pre-cancerous lesions, safely. In particular, thepresent invention is a method for selecting compounds that can be usedto treat and prevent neoplasia, including precancerous lesions. Thecompounds so identified can have minimal side effects attributable toCOX inhibition and other non-specific interactions associated withconventional chemotherapeutics. The compounds of interest can be testedby exposing the novel PDE described above to the compounds, and if acompound inhibits this novel PDE, the compound is then firther evaluated(e.g., in vitro or in vivo animal or human testing models or trials) forits anti-neoplastic properties.

One aspect of this invention, therefore, involves a screening/selectionmethod to identify a compound effective for treating neoplasia thatincludes ascertaining the compound's inhibition of this novel PDE and/orPDE2 and its inhibition of COX. Preferably, the screening and selectionmethods of this invention further include determining whether thecompound inhibits the growth of tumor cells in vitro or in vivo.

By selecting compounds in this fashion, potentially beneficial andimproved compounds for treating neoplasia can be identified more rapidlyand with greater precision than possible in the past for the purposes ofdeveloping pharmaceutical compositions and therapeutically treatingneoplasia. Further benefits will be apparent from the following detaileddescription.

This invention also includes pharmaceutical compositions containing suchcompounds, as well as therapeutic methods involving such compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from SW480 neoplastic cells, as assayed from the eluent from aDEAE-Trisacryl M column.

FIG. 2 is a graph of cGMP activities of the reloaded cGMPphosphodiesterases obtained from SW480 neoplastic cells, as assayed fromthe eluent from a DEAE-Trisacryl M column.

FIG. 3 is a graph of the kinetic behavior of the novel PDE of thisinvention.

FIG. 4 illustrates the effect of the sulfide derivative of sulindac andthe sulfone derivative of sulindac (a.k.a. exisulind) on purifiedcyclooxygenase activity.

FIG. 5 illustrates the effects of test compounds B and E on COXinhibition.

FIG. 6 illustrates the inhibitory effects of sulindac sulfide andexisulind on PDE4 and PDE5 Purified from cultured tumor cells.

FIG. 7 illustrates the effects of sulindac sulfide on cyclic nucleotidelevels in HT-29 cells.

FIG. 8 illustrates the phosphodiesterase inhibitory activity of compoundB.

FIG. 9 illustrates the phosphodiesterase inhibitory activity of compoundE.

FIG. 10 illustrates the effects of sulindac sulfide and exisulind onapoptosis and necrosis of HT-29 cells.

FIG. 11. illustrates the effects of sulindac sulfide and exisulind onHT-29 cell growth inhibition and apoptosis induction as determined byDNA fragmentation.

FIG. 12 illustrates the apoptosis-inducing properties of compound E.

FIG. 13 illustrates the apoptosis-inducing properties of compound B.

FIG. 14 illustrates the effects of sulindac sulfide and exisulind ontumor cell growth.

FIG. 15 illustrates the growth inhibitory and apoptosis-inducingactivity of sulindac sulfide and control (DMSO).

FIG. 16 illustrates the growth inhibitory activity of compound E.

FIG. 17 illustrates the inhibition of pre-malignant, neoplastic lesionsin mouse mammary gland organ culture by sulindac metabolites.

FIG. 18A is a SDS protein gel of SW480 cell lysates from drug-treatedcell lysates in the absence of added cGMP, where cells were treated inculture for 48 hours with DMSO (0.03%, lanes 1 and 2), exisulind (200,400 and 600 μM; lanes 3, 4, 5) and E4021 (0.1, 1 and 10 μM, lanes 6, 7,8).

FIG. 18B is a SDS (X-ray film exposure) gel PKG assay of SW480 celllysates from drug-treated cell lysates in the presence of added cGMP,where cells were treated in culture for 48 hours with DMSO (0.03%, lane2), exisulind (200, 400 and 600 μM; lanes 3, 4, 5) and E4021 (0.1, 1 and10 μM, lanes 6, 7, 8).

FIG. 19 is a bar graph of the results of Western blot experiments of theeffects of exisulind on β-catenin and PKG levels in neoplastic cellsrelative to control.

FIG. 20 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from HTB-26 neoplastic cells, as assayed from the eluent from aDEAE-Trisacryl M column.

FIG. 21 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from HTB-26 neoplastic cells, as assayed from the eluent from aDEAE-Trisacryl M column with low and high substrate concentration.

FIG. 22 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from LnCAP neoplastic cells, as assayed from the eluent from aDEAE-Trisacryl M column.

FIG. 23 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from LnCAP neoplastic cells, as assayed from the eluent from aDEAE-Trisacryl M column with low and high substrate concentration.

FIG. 24 is a bar graph illustrating the specificity binding of thenon-catalytic cGMP binding sites of PDE5 for cyclic nucleotide analogsand selected PDE5 inhibitors.

FIG. 25 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from SW480 neoplastic cells, as assayed from the eluent from aDEAE-Trisacryl M column using ethylene glycol in the buffer.

FIG. 26 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from SW480 neoplastic cells grown in roller bottles, as assayedfrom the eluent from a DEAE-Trisacryl M column.

FIG. 27A shows a time-dependent increase in the amount ofhistone-associated fragmented DNA in LNCaP cell cultures followingtreatment with 50 μM Compound I.

FIG. 27B shows the course of treatment of PrEC prostate cells withCompound I (50 μM) that did not affect DNA fragmentation for up to 4days of treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. The Novel cGMP-Specific Phosphodiesterase And PDE2 From NeoplasticCells

A. The Isolation Of The Novel PDE Conformation

The isolated cGMP-specific phosphodiesterase (which appears to be anovel conformation of PDE2) was first prepared from the human carcinomacell line commonly referred to as SW480 available from the AmericanTissue Type Collection in Rockville, Md., U.S.A. SW480 is a human coloncancer cell line that originated from moderately differentiatedepithelial adenocarcinoma. As discussed below, a similar conformationhas also been isolated from neoplasias of the breast (i.e., HTB-26 cellline) and prostate (i.e., LNCAP cell line).

By “isolated” we mean (as is understood in the art) not only isolatedfrom neoplastic cells, but also made by recombinant methods (e.g.,expressed in a bacterial or other non-human host vector cell lines).However, we presently believe isolation from the human neoplastic cellline is preferable since we believe that the target protein so isolatedhas a structure (i.e., a conformation or topography) that is closer to,if not identical with, one of the native conformations in the neoplasticcell as possible. This conformation assists in the selection ofanti-neoplastic compounds that will inhibit the target enzyme(s) invivo.

The novel PDE activity was first found in SW480 colon cancer cell lines.To isolate the novel phosphodiesterase from SW480, approximately fourhundred million SW480 cells were grown to confluence in and were scrapedfrom 150 cm² tissue culture dishes after two washes with 10 mL cold PBSand pelleted by centrifugation. The cells were re-suspended inhomogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5mM MgAc₂, 0.1 mM EDTA, 0.8% Triton-100, 10 μM benzamidine, 10 μM TLCK,2000 U/mL aprotinin, 2 μM leupeptin, 2 μM pepstatin A) and homogenizedon an ice bath using a polytron tissumizer (three times, 20seconds/pulse). The homogenized material was centrifuged at 105,000 gfor 60 minutes at 4° C. in a Beckman L8 ultracentrifuge, and thesupernatant was diluted with TMPI-EDTA (60 mL) and applied to a10-milliliter DEAE-Trisacryl M column pre-equilibrated with TMPI-EDTAbuffer. The loaded column was washed with 60 mL of TM-EDTA, and PDEactivities were eluted with a 120 mL linear gradient of NaOAC (0-0.5 M)in M-EDTA, at a flow rate of 0.95 mL/minute, 1.4 mL/fraction. Eightyfractions were collected and assayed for cGMP hydrolysis immediately(i.e. within minutes). FIG. 1. shows the column's elution profile,revealing two initial peaks of cGMP PDE activity, peaks A and B, whichwere eluted by 40-50 mM and 70-80 mM NaOAC, respectively. As explainedbelow, peak A is PDE5, whereas peak B is a novel cGMP-specificphosphodiesterase activity.

Cyclic nucleotide PDE activity of each fraction was determined using themodified two-step radio-isotopic method of Thompson et al. (Thompson W.J., et al., Adv. Cyclic Nucleotide Res. 10: 69-92, 1979), as furtherdescribed below. The reaction was in 400 μl containing Tris-HCl (40 mM;pH 8.0), MgCl₂ (5 mM), 2-mercaptoethanol (4 mM), bovine serum albumin(30 μg), cGMP (0.25μM-5 μM) with constant tritiated substrate (200,000cpm). The incubation time was adjusted to give less than 15% hydrolysis.The mixture was incubated at 30° C. followed by boiling for 45 secondsto stop the reaction. Then, the mixture was cooled, snake venom (50 μg)added, and the mixture was incubated at 30° C. for 10 minutes. MeOH (1mL) was added to stop the reaction, and the mixture was transferred toan anion-exchange column (Dowex 1-X8, 0.25 mL resin). The eluent wascombined with a second mL of MeOH, applied to the resin, and afteradding 6 mL scintillation fluid, tritium activity was measured using aBeckman LS 6500 for one minute.

To fractionate the cGMP hydrolytic activities of peaks A and B further,fractions 15 to 30 of the original 80 were reloaded onto theDEAE-Trisacryl M column and eluted with a linear gradient of NaOAC(0-0.5 M) in TM-EDTA. Fractions were again immediately assayed for cGMPhydrolysis (using the procedure described above with 0.2, 2, 5 μMsubstrate), the results of which are graphically presented in FIG. 2.One observation about peak B illustrated in FIG. 2 is that increasingsubstrate concentration of cGMP dramatically enhanced activity whencontrasted to peak A. While this observation is consistent with itsbeing a PDE2, the fact that the enzyme characterized in FIG. 2 iscGMP-specific (see below) suggests that it has a novel conformationcompared to the classic PDE2 reported in the literature. Peak A activityshows apparent substrate saturation of high affinity catalytic sites.

B. The Isolation of Classic PDE2 From SW480

Two methods were found that allowed “peak B” to be isolated from SW480so that the enzyme had the classical PDE2 activity (i.e. was notcGMP-specific, but was cGMP stimulated). The first method involvedgrowing the SW480 in 850 cm² Corning roller bottles instead of 150 cm²tissue culture flasks. SW480 were grown in roller bottles at 0.5 rpmwith each bottle containing 200 mL of RPMI 1640, 2 mM glutamine, and 25mM HEPES. Cells were harvested by the following procedure. PBS media waswarmed to 37° C. for at least 15 minutes. 200 mL of 5% FBS/RPMI 1640complete media is prepared and 5 mL of glutamine were added. 5 mL ofantibiotic/antimycotic were also added. 70 mL of the PBS solution wasadded to 10 mL of 4×Pancreatin. The mixture was maintained at roomtemperature. The media was removed and the flask was rinsed with 4 mL ofPBS being sure the bottom of the flask was covered. All solution wasremoved with a pipet. 4 mL of diluted Pancreatin was added to the flask,and the flask was swished to cover its bottom. The flask was incubatedat 37° C. for 8-10 minutes. After the incubation, the flask was quicklychecked under an inverted microscope to make sure all cells wererounded. The flask was hit carefully on its side several times to helpdetach cells. 10 mL of cold complete media were added to the flask tostop the Pancreatin proteolysis. The solution was swirled over thebottom to collect the cells. The media was removed using a 25 mL pipet,and the cells placed in 50 mL centrifuge tubes on ice. The tubes werespun at 1000 rpm at 4° C. for 5 minutes in a clinical centrifuge topellet cells. The supernatant was poured off and each pellet frozen onliquid nitrogen for 15 seconds. The harvested cells can be stored in a−70° C. freezer.

The PDEs from the harvested SW480 cells were isolated using a FPLCprocedure. A Pharmacia AKTA FPLC was used to control sample loading andelution on an 18 mL DEAE TrisAcryl M column. About 600 million cells ofSW480 were used for the profiles. After re-suspending cells inhomogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5mM MgAc₂, 0.1 mM EDTA, 0.8% Triton-100, 10 μM benzamidine, 10 μM TLCK,2000 U/mL aprotinin, 2 μM leupeptin, 2 μM pepstatin A), samples weremanually homogenized. FPLC buffer A was 8 mM TRIS-acetate, 5 mM Mgacetate, 0.1 mM EDTA, pH 7.5 and buffer B was 8 mM TRIS-acetate, 5 mM Mgacetate, 0.1 mM EDTA, 1 M Na acetate, pH 7.5. Supernatants were loadedonto the column at 1 mL per minute, followed by a wash with 60 mL bufferA at 1 mL per minute. A gradient was run from 0-15% buffer B in 60 mL,15-50% buffer B in 60 mL, and 50-100% buffer B in 16 mL. During thegradient, 1.5 mL fractions were collected.

The profile obtained was similar (FIG. 26) to the profile for the novelPDE activity (see, e.g., FIG. 1) obtained above, except that Peak Bisolated in this manner showed cAMP hydrolytic activity at 0.25 μMsubstrate that could be activated 2-3 fold by 5 μM cGMP.

A second method used to isolate classic PDE2 from SW480 was done using anon-FPLC DEAE column procedure described above (see Section IA) with themodification that the buffers contained 30% ethylene glycol, 10 mM TLCKand 3.6 mM β-mercaptoethanol. The addition of these reagents to thebuffers causes a shift in the elution profile (see FIG. 25) from low tohigh sodium acetate so that peak A moves from 40 to 150 mM, peak B from75 to 280 mM and peak C from 200 to 500 mM Na acetate (see FIG. 25).Peak B in FIG. 25 was assayed with 2 μM cAMP substrate and showed atwo-fold activation by 5 μM cGMP (see Figure −Y). The selective PDE2inhibitor EHNA inhibited 2 μM cGMP PDE activity in this Peak B with anIC₅₀ of 1.6 μM and inhibited 2.0 μM cAMP PDE activity in Peak B with anIC₅₀ of 3.8 μM (and IC₅₀ of 2.5 μM with addition of 10 μM rolipram).

C. cGMP-Specificity of PDE Peak A and The Novel Peak B Activity

Each fraction from the DEAE column from Section IA was also assayed forcGMP-hydrolysis activity (0.25 μM cGMP) in the presence or absence ofCa⁺⁺, or Ca⁺⁺-CaM and/or EGTA and for cAMP (0.25 μM cAMP) hydrolysisactivity in the presence or absence of 5 μM cGMP. Neither PDE peak A andpeak B (fractions 5-22; see FIG. 1) hydrolyzed cAMP significantly,establishing that neither had the activity of a classic cAMP-hydrolyzingfamily of PDE (i.e. a PDE 1, 2, 3).

Ca⁺⁺(with or without calmodulin) failed to activate either cAMP or cGMPhydrolysis activity of either peak A or B, and cGMP failed to activateor inhibit cAMP hydrolysis. Such results establish that peaks A and Bconstitute cGMP-specific PDE activities but not classic or previouslyknown PDE1, PDE2, PDE3 or PDE4 activities.

For the novel PDE peak B, as discussed below, cyclic GMP activated thecGMP hydrolytic activity of the enzyme, but did not activate any cAMPhydrolytic activity (in contrast with the Peak B from Section IB above).reveals that the novel PDE peak B—the novel phosphodiesterase of thisinvention—is not a cGMP-stimulated cAMP hydrolysis (“cGS”) or among theclassic or previously known PDE2 family activities because the knownisoforms of PDE2 hydrolyze both cGMP and cAMP.

D. Peak A Is A Classic PDE5, But The Novel Peak B—A New cGMPSpecificPDE-Is Not

To characterize any PDE isoform, kinetic behavior and substratepreference should be assessed.

Peak A showed typical “PDE5” characteristics. For example, the k_(m) ofthe enzyme for cGMP was 1.07 μM, and Vmax was 0.16 nmol/min/mg. Inaddition, as discussed below, zaprinast (IC₅₀=1.37 μM) and E4021 (IC₅₀323 nM) and sildenafil inhibited activity of peak A. Further, zaprinastshowed inhibition for cGMP hydrolysis activity of peak A, consistentwith results reported in the literature.

PDE Peak B from Section IA showed considerably different kineticproperties as compared to PDE peak A. For example, in Eadie-Hofsteeplots of Peak A, cyclic GMP hydrolysis shows single line with negativeslope with increasing substrate concentrations, indicative ofMichaelis-Menten kinetic behavior. Peak B, however, shows the novelproperty for cGMP hydrolysis in the absence of cAMP of a decreasing(apparent K_(m)=8.4), then increasing slope (K_(m)<1) of Eadie-Hotfsteeplots with increasing cGMP substrate (see, FIG. 3). Thus, thisestablishes Peak B's submicromolar affinity for cGMP (i.e., whereK_(m)<1).

Consistent with the kinetic studies (i.e., FIG. 3) andpositive-cooperative kinetic behavior in the presence of cGMP substrate,was the increased cGMP hydrolytic activity in the presence of increasingconcentrations of cGMP substrate. This was discovered by comparing 0.25μM, 2 μM and 5 μM concentrations of cGMP in the presence of PDE peak Bafter a second DEAE separation to rule out cAMP hydrolysis and to ruleout this new enzyme being a previously identified PDE5. Higher cGMPconcentrations evoked disproportionately greater cGMP hydrolysis withPDE peak B, as shown in FIG. 2.

These observations suggest that cGMP binding to the peak B enzyme causesa conformational change in the enzyme. This confirms the advantage ofusing the native enzyme from neoplastic cells, but this invention is notlimited to the native form of the enzyme having the characteristics setforth above.

E. Zaprinast- and Sildenafil-Insensitivity of PDE Peak B Relative toPeak A, and Their Effects on Other PDE Inhibitors

Different PDE inhibitors were studied using twelve concentrations ofdrug from 0.01 to 100 μM and substrate concentration of 0.25 μM ³H-cGMP.IC₅₀ values were calculated with variable slope, sigmoidal curve fitsusing Prism 2.01 (GraphPad). The results are shown in Table 1. Whilecompounds E4021 and zaprinast inhibited peak A, (with high affinities)IC₅₀ values calculated against the novel PDE activity in peak B (SectionIA) are significantly increased (>50 fold). This confirms that peak A isa PDE5. These data further illustrate that the novel PDE activity ofthis invention is, for all practical purposes, zaprinast-insensitive andE4021-insensitive.

TABLE 1 Comparison of PDE Inhibitors Against Peak A and Section IA PeakB (cGMP Hydrolysis) Ratio (IC₅₀ PDE Family IC₅₀ IC₅₀ PeakA/ CompoundInhibitor Peak A (μM) Peak B (μM) Peak B) E4021 5 0.003 8.4 0.0004Zaprinast 5 1.4 >30 <0.05 Compound E 5 and others 0.38 0.37 1.0 Sulindacsulfide 5 and others 50 50 1.0 Vinpocetine 1 >100 >100 EHNA 2,5 >100 3.7Indolidan 3 31 >100 <0.31 Rolipram 4 >100 >100 Sildenafil 5 .0003 >10<.00003

By contrast, sulindac sulfide and Compound E and competitvely inhibitedboth peaks A and B phosphodiesterases at the same potency (IC₅₀=0.38 μMfor PDE peak A; 0.37 μM for PDE peak B).

There is significance for the treatment in the fact that peak B (eitherform of it) is zaprinast-insensitive whereas peaks A and B are bothsensitive to sulindac sulfide and Compound E. We have tested zaprinast,E4021 and sildenafil to ascertain whether they induce apoptosis orinhibit the growth of neoplastic cells, and have done the same forCompound E. As explained below, zaprinast by itself does not havesignificant apoptosis-inducing or growth-inhibiting properties, whereassulindac sulfide and Compound E are precisely the opposite. In otherwords, the ability of a compound to inhibit both PDE peaks A and Bcorrelates with its ability to induce apoptosis in neoplastic cells,whereas if a compound (e.g., zaprinast) has specificity for PDE peak Aonly, that compound will not by itself induce apoptosis.

F. Insensitivity of The Novel PDE Peak B To Incubation WithcGMP-Dependent Protein Kinase G

Further differences between PDE peak A and the novel peak B (Section IA)were observed in their respective cGMP-hydrolytic activities in thepresence of varying concentrations of cGMP-dependent protein kinase G(which phosphorylates typical PDE5). Specifically, peak A and peak Bfractions from Section IA were incubated with different concentrationsof protein kinase G at 30° C. for 30 minutes. Cyclic GMP hydrolysis ofboth peaks has assayed after phosphorylation was attempted. Consistentwith previously published information about PDE5, Peak A showedincreasing cGMP hydrolysis activity in response to protein kinase Gincubation, indicating that Peak A was phosphorylated. Peak B wasunchanged, however (i.e., was not phosphorylated and insensitive toincubation with cGMP-dependent protein kinase G). These data areconsistent with Peak A being an isoform consistent with the known PDE5family and Peak B from Section IA being a novel cGMP-specific PDEactivity.

G. Novel Peak B In Prostate and Breast Cancer Cell Lines

The novel Peak B was also isolated from two other neoplastic cell lines,a breast cancer cell line, HTB-26 and a prostate cancer cell line, LnCAPby a procedure similar to the one above used to isolate it from SW480.The protocol was modified in several respects. To provide even greaterreproducibility to allow comparison of different cell lines, a PharmaciaAKTA FPLC was used to control sample loading and elution on an 18 mLDEAE TrisAcryl M column. SW840 was run by this same procedure multipletimes to provide a reference of peak B. 200-400 million cells of SW480were used for the profiles. 70 million cells of LnCAP were used for aprofile (see FIGS. 22 and 23), and in a separate experiment 32 millioncells of HTB-26 were used for a profile (see FIGS. 20 and 21). Afterre-suspending cells in homogenization buffer, samples were manuallyhomogenized. FPLC buffer A was 8 mM TRIS-acetate, 5 mM Mg acetate, 0.1mM EDTA, pH 7.5 and buffer B was 8 mM TRIS-acetate, 5 mM Mg acetate, 0.1mM EDTA, 1 M Na acetate, pH 7.5. Supernatants were loaded onto thecolumn at 1 mL per minute, followed by a wash with 60 mL buffer A at 1mL per minute. A gradient was run from 0-15% buffer B in 60 mL, 15-50%buffer B in 60 mL, and 50-100% buffer B in 16 mL. During the gradient1.5 mL fractions were collected. Peaks of cGMP PDE activity elutedaround fraction 65 that was at 400 mM Na acetate (see FIGS. 20-23). Thisactivity was measured at 0.25 μM cGMP (indicating submicromolar affinityfor cGMP). Rolipram, a PDE4-specific drug, inhibited most of the cAMPPDE activity (i.e. the cAMP activity was due to PDE4), indicating thatthe peak B's cGMP activity were specific for cGMP over cAMP. All threepeak B's (from SW480, HTB-26, and LnCAP) did not show stimulation withcalcium/calmodulin and were resistant to 100 nM E4021, a specificPDE5-specific inhibitor like zaprinast (see FIGS. 20 and 22). The peakB's also showed a dramatic increase in activity when substrate wasincreased from 0.25 μM to 5 μM cGMP (suggesting positively cooperativekinetics) (see FIGS. 21 and 23). Also, the three peaks show similarinhibition by exisulind and Compound I, below.

II. Protein Kinase G and β-Catenin Involvement—In General

A series of experiments were performed to ascertain what effect, if any,an anti-neoplastic cGMP-specific PDE inhibitor such as exisulind had oncGMP-dependent protein kinase G (“PKG”) in neoplastic cells containingeither the adenomatous polyposis coli gene (“APC gene”) defect or adefect in the gene coding for β-catenin. As explained below, such aninhibitor causes an elevation in PKG activity in such neoplastic cells.That increase in activity was not only due to increased activation ofPKG in cells containing either defect, but also to increased expressionof PKG in cells containing the APC defect. In addition, when PKG fromneoplastic cells with either defect is immunoprecipitated, itprecipitates with β-catenin.

β-catenin has been implicated in a variety of different cancers becauseresearchers have found high levels of it in patients with neoplasiascontaining mutations in the APC tumor-suppressing gene. People withmutations in this gene at birth often develop thousands of small tumorsin the lining of their colon. When it functions properly, the APC genecodes for a normal APC protein that is believed to bind to and regulateβ-catenin. Thus, the discovery that PKG in neoplastic cells containingeither the APC gene defect or the β-catenin defect is bound to β-cateninindeed strongly implicates PKG in one of the major cellular pathwaysthat leads to cancer. In addition, because of the relationship betweencGMP-specific inhibition and PKG elevation upon treatment with SAANDslinks cGMP to the PKG/β-catenin/APC defect in such cells.

This latter link is further buttressed by the observation that β-cateninitself is reduced when neoplastic cells containing the APC defect or theβ-catenin defect are exposed to a SAAND. This reduction in β-catenin isinitiated by PKG itself. PKG phosphorylates β-catenin—which is anothernovel observation associated with this invention. The phosphorylation ofβ-catenin allows β-catenin to be degraded by ubiquitin-proteasomalsystem.

This phosphorylation of β-catenin by PKG is important in neoplasticcells because it circumvents the effect of the APC and β-cateninmutations. The mutated APC protein affects the binding of the β-cateninbound to the mutant APC protein, which change in binding has heretoforebeen thought to prevent the phosphorylation of β-catenin by GSK-3 bkinase. In the case of mutant β-catenin, an elevation of PKG activityalso allows the mutant β-catenin to be phosphorylated. By elevating PKGactivity in neoplasia with cGMP-PDE inhibition allows for β-cateninphosphorylation leading to its degradation) in neoplastic cellscontaining either type of mutation.

In short, these findings not only lead to new pharmaceutical screeningmethods to identify further SAAND candidate compounds, but also buttressthe role of cGMP-specific PDE inhibition in therapeutic approaches toneoplasia. This observation may also explain the unexpectedly broadrange of neoplasias SAANDs can inhibit since both neoplasia with andwithout the APC defect can be treated, as explained above.

III. Screening Pharmaceutical Compositions Usins The PDEs

A. In General

The novel PDE of this invention and PDE2 are useful with or without PDE5to identify compounds that can be used to treat or prevent neoplasms,and that are not characterized by serious side effects.

Cancer and precancer may be thought of as diseases that involveunregulated cell growth. Cell growth involves a number of differentfactors. One factor is how rapidly cells proliferate, and anotherinvolves how rapidly cells die. Cells can die either by necrosis orapoptosis depending on the type of environmental stimuli. Celldifferentiation is yet another factor that influences tumor growthkinetics. Resolving which of the many aspects of cell growth is affectedby a compound is important to the discovery of a relevant target forpharmaceutical therapy. Screening assays based on this technology can becombined with other tests to select compounds that have growthinhibiting and pro-apoptotic activity.

This invention is the product of several important discoveries. First,the present inventors discovered that desirable inhibitors of tumor cellgrowth induce premature death of cancer cells by apoptosis (see, Piazza,G. A., et al., Cancer Research, 55(14), 3110-16, 1995). Second, severalof the present inventors unexpectedly discovered compounds thatselectively induce apoptosis without substantial COX inhibition alsoinhibit PDE5. In particular, and contrary to leading scientific studies,desirable compounds for treating neoplastic lesions inhibit PDE5 (EC3.1.4.17). PDE5 is one of at least ten gene families ofphosphodiesterase. PDE5 and the novel PDE of this invention are uniquein that they selectively degrade cyclic GMP and not cAMP, while theother families of PDE selectively degrade/hydrolyze cAMP and not cGMP ornon-selectively degrade both cGMP and cAMP. Preferably, desirablecompounds used to treat neoplasia do not substantially inhibitnon-selective or cAMP degrading phosphodiesterase types.

B. COX Screening

A preferred embodiment of the present invention involves determining thecyclooxygenase inhibition activity of a given compound, and determiningthe cGMP specific PDE inhibitory activity of the compound. The testcompounds are assessed for their ability to treat neoplastic lesionseither directly or indirectly by comparing their activities againstknown compounds useful for treating neoplastic lesions. A standardcompound that is known to be effective for treating neoplastic lesionswithout causing gastric irritation is5-fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetic acid(“exisulind”). Other useful compounds for comparative purposes includethose that are known to inhibit COX, such as indomethacin and thesulfide metabolite of sulindac:5-fluoro-2-methyl-1-(p-methylsulfinylbenzylidene)-3-indenyl acid(“sulindac sulfide”). Other useful compounds for comparative purposesinclude those that are known to inhibit (cGMP-specific PDEs, such as1-(3-chloroanilino)-4-phenyphthalazine (“MY5445”).

As used herein, the term “precancerous lesion” includes syndromesrepresented by abnormal neoplastic, including dysplastic, changes oftissue. Examples include dysplastic growths in colonic, breast, prostateor lung tissues, or conditions such as dysplastic nevus syndrome, aprecursor to malignant melanoma of the skin. Examples also include, inaddition to dysplastic nevus syndromes, polyposis syndromes, colonicpolyps, precancerous lesions of the cervix (i.e., cervical dysplasia),esophagus, lung, prostatic dysplasia, prostatic intraneoplasia, breastand/or skin and related conditions (e.g., actinic keratosis), whetherthe lesions are clinically identifiable or not.

As used herein, the terms “carcinoma” or “cancer” refers to lesionswhich are cancerous. Examples include malignant melanomas, breastcancer, prostate cancer and colon cancer. As used herein, the terms“neoplasia” and “neoplasms” refer to both cancerous and pre-cancerouslesions.

As used herein, the abbreviation PG represents prostaglandin; PSrepresents prostaglandin synthetase; PGE₂ represents prostaglandin E₂;PDE represents phosphodiesterase; COX represents cyclooxygenase; cyclicnucleotide, RIA represents—radioimmunoassay.

COX inhibition by a compound can be determined by either of two methods.One method involves measuring PGE₂ secretion by intact HL-60 cellsfollowing exposure to the compound being screened. The other methodinvolves measuring the activity of purified cyclooxygenases (COXs) inthe presence of the compound. Both methods involve protocols previouslydescribed in the literature, but preferred protocols are set forthbelow.

Compounds can be evaluated to determine whether they inhibit theproduction of prostaglandin E₂ (“PGE₂”), by measuring PGE₂. Using anenzyme immunoassay (EIA) kit for PGE₂, such as commercially availablefrom Amersham, Arlington Heights, Ill. U.S.A. Suitable cells includethose that make an abundance of PG, such as HL-60 cells. HL-60 cells arehuman promyelocytes that are differentiated with DMSO into maturegranulocytes (see, Collins, S. J., Ruscetti, F. W., Gallagher, R. E. andGallo, R. C., “Normal Functional Characteristics of Cultured HumanPromyelocytic Leukemia Cells (HL-60) After Induction of DifferentiationBy Dimethylsulfoxide”, J Exp. Med, 149:969-974, 1979). Thesedifferentiated cells produce PGE₂ after stimulation with a calciumionophore, A23187 (see, Kargman, S., Prasit, P. and Evans, J. F.,“Translocation of HL-60 Cell 5-Lipoxygenase”, J. Biol. Chem., 266:23745-23752, 1991). HL-60 are available from the ATCC (ATCC:CCL240).They can be grown in a RPMI 1640 medium supplemented with 20%heat-inactivated fetal bovine serum, 50 U/mL penicillin and 50 μg/mLstreptomycin in an atmosphere of 5% CO₂ at 37° C. To induce myeloiddifferentiation, cells are exposed to 1.3% DMSO for 9 days and thenwashed and resuspended in Dulbecco's phosphate-buffered saline at aconcentration of 3×10⁶ cells/mL.

The differentiated HL-60 cells (3×10⁶ cells/mL) are incubated for 15minutes at 37° C. in the presence of the compounds tested at the desiredconcentration. Cells are then stimulated by A23187 (5×10⁶ M) for 15minutes. PGE₂ secreted into the external medium is measured as describedabove.

As indicated above, a second method to assess COX inhibition of acompound is to measure the COX activity in the presence of a testcompound. Two different forms of cyclooxygenase (COX-I and COX-2) havebeen reported in the literature to regulate prostaglandin synthesis.COX-2 represents the inducible form of COX while COX-I represents aconstitutive form. COX-I activity can be measured using the methoddescribed by Mitchell et al. (“Selectivity of NonsteroidalAnti-inflammatory Drugs as Inhibitors of Constitutive and InducibleCyclooxygenase,” Proc. Natl. Acad. Sci. USA., 90:11693-11697, 1993,which is incorporated herein by reference) using COX-I purified from ramseminal vesicles as described by Boopathy & Balasubramanian,“Purification And Characterization Of Sheep Platelet Cyclooxygenase”(Biochem. J., 239:371-377, 1988, which is incorporated herein byreference). COX-2 activity can be measured using COX-2 Purified fromsheep placenta as described by Mitchell et al., 1993, supra.

The cyclooxygenase inhibitory activity of a drug can be determined bymethods known in the art. For example, Boopathy & Balasubramanian, 1988,supra, described a procedure in which prostaglandin H synthase 1 (CaymanChemical, Ann Arbor, Mich.) is incubated at 37° C. for 20 minutes with100 μM arachidonic acid (Sigma Chemical Co.), cofactors (such as 1.0 mMglutathione, 1.0 mM hydroquinone, 0.625 μM hemoglobin and 1.25 mM CaCl₂in 100 mM Tris-HCl, pH 7.4) and the drug to be tested. Followingincubation, the reaction can be terminated with trichloroacetic acid.After stopping the reaction by adding thiobarbituric acid andmalonaldehyde, enzymatic activity can then be measuredspectrophotometrically at 530 nm.

Obviously, a compound that exhibits a lower COX-I or COX-2 inhibitoryactivity in relation to its greater combined PDE5/novel PDE/PDE2inhibitory activities may be a desirable compound.

The amount of COX inhibition is determined by comparing the activity ofthe cyclooxygenase in the presence and absence of the test compound.Residual (i.e., less than about 25%) or no COX inhibitory activity at aconcentration of about 100 μM is indicative that the compound should beevaluated further for usefulness for treating neoplasia.

C. Determining Phosphodiesterase Inhibition Activity

Compounds can be screened for inhibitory effect on the activity of thenovel phosphodiesterase of this invention using either the enzymeisolated as described above, a recombinant version, or using the novelPDE and/or PDE2 together with PDE5. Alternatively, cyclic nucleotidelevels in whole cells are measured by RIA and compared to untreated andzaprinast-treated cells.

Phosphodiesterase activity can be determined using methods known in theart, such as a method using radioactive ³H cyclic GMP (cGMP)(cyclic3′,5′-guanosine monophosphate) as the substrate for the PDE enzyme.(Thompson, W. J., Teraski, W. L., Epstein, P. M., Strada, S. J.,Advances in Cyclic Nucleotide Research, 10:69-92, 1979, which isincorporated herein by reference). In brief, a solution of defmedsubstrate ³H-cGMP specific activity (0.2 μM; 100,000 cpm; containing 40mM Tris30 HCl (pH 8.0), 5 mM MgCl₂ and 1 mg/mL BSA) is mixed with thedrug to be tested in a total volume of 400 μl. The mixture is incubatedat 30° C. for 10 minutes with isolated PDE of this invention. Reactionsare terminated, for example, by boiling the reaction mixture for 75seconds. After cooling on ice, 100 μl of 0.5 mg/mL snake venom (O.Hannah venom available from Sigma) is added and incubated for 10 minutesat 30° C. This reaction is then terminated by the addition of analcohol, e.g. 1 mL of 100% methanol. Assay samples are applied to 1 mLDowex 1-X8 column; and washed with 1 mL of 100% methanol. The amount ofradioactivity in the breakthrough and the wash from the column iscombined and measured with a scintillation counter. The degree ofphosphodiesterase inhibition is determined by calculating the amount ofradioactivity in drug-treated reactions and comparing against a controlsample (a reaction mixture lacking the tested compound but with drugsolvent).

Alternatively, the ability of desirable compounds to inhibit thephosphodiesterases of this invention is reflected by an increase in cGMPin neoplastic cells exposed to a compound being screened. The amount ofPDE activity can be determined by assaying for the amount of cyclic GMPin the extract of treated cells using radioimmunoassay (RIA). In thisprocedure, HT-29 or SW-480 cells are plated and grown to confluency. Asindicated above, SW-480 contains both PDE5 and the novel PDE of thisinvention, so when PDE activity is evaluated in this fashion, a combinedcGMP hydrolytic activity is assayed simultaneously. The test compound isthen incubated with the cell culture at a concentration of compoundbetween about 200 μM to about 200 μM. About 24 to 48 hours thereafter,the culture media is removed from the cells, and the cells aresolubilized. The reaction is stopped by using 0.2N HCl/50% MeOH. Asample is removed for protein assay. Cyclic GMP is purified from theacid/alcohol extracts of cells using anion-exchange chromatography, suchas a Dowex column. The cGMP is dried, acetylated according to publishedprocedures, such as using acetic anhydride in triethylamine, (Steiner,A. L., Parker, C. W., Kipnis, D. M., J. Biol. Chem., 247(4):1106-13,1971, which is incorporated herein by reference). The acetylated cGMP isquantitated using radioimmunoassay procedures (Harper, J., Brooker, G.,Advances in Nucleotide Research, 10:1-33, 1979, which is incorporatedherein by reference). lodinated ligands (tyrosine methyl ester) ofderivatized cyclic GMP are incubated with standards or unknowns in thepresence of antisera and appropriate buffers. Antiserum may be producedusing cyclic nucleotide-haptene directed techniques. The antiserum isfrom sheep injected with succinyl-cGMP-albumin conjugates and diluted1/20,000. Dose-interpolation and error analysis from standard curves areapplied as described previously (Seibert, A. F., Thompson, W. J.,Taylor, A., Wilboum, W. H., Barnard, J. and Haynes, J., J AppliedPhysiol., 72:389-395, 1992, which is incorporated herein by reference).

In addition, the culture media may be acidified, frozen (-70° C.) andalso analyzed for cGMP and cAMP.

In addition to observing increases in the content of cGMP in neoplasticcells caused by desirable compounds, decreases in content of cAMP havealso been observed. It has been observed that a particularly desirablecompound (i.e., one that selectively induces apoptosis in neoplasticcells, but not substantially in normal cells) follows a time courseconsistent with cGMP-specific PDE inhibition as one initial actionresulting in an increased cGMP content within minutes. Secondarily,treatment of neoplastic cells with a desirable anti-neoplastic compoundleads to decreased cAMP content within 24 hours. The intracellulartargets of drug actions are being studied further, but current datasupport the concept that the initial rise in cGMP content and thesubsequent fall in cAMP content precede apoptosis in neoplastic cellsexposed to desirable compounds.

The change in the ratio of the two cyclic nucleotides may be a moreaccurate tool for evaluating desirable cGMP-specific phosphodiesteraseinhibition activity of test compounds, rather than measuring only theabsolute value of cGMP, only cGMPspecific phosphodiesterase inhibition,or only the level of cGMP hydrolysis. In neoplastic cells not treatedwith anti-neoplastic compounds, the ratio of cGMP content/cAMP contentis in the 0.03-0.05 range (i.e., 300-500 fmol/mg protein cGMP contentover 6000-8000 fmol/mg protein cAMP content). After exposure todesirable anti-neoplastic compounds, that ratio increases several fold(preferably at least about a three-fold increase) as the result of aninitial increase in cyclic GMP and the later decrease in cyclic AMP.

Specifically, it has been observed that particularly desirable compoundsachieve an initial increase in cGMP content in treated neoplastic cellsto a level of cGMP greater than about 500 fmol/mg protein. In addition,particularly desirable compounds cause the later decrease in cAMPcontent in treated neoplastic cells to a level of cAMP less than about4000 fmol/mg protein.

To determine the content of cyclic AMP, radioimmunoassay techniquessimilar to those described above for cGMP are used. Basically, cyclicnucleotides are purified from acid/alcohol extracts of cells usinganion-exchange chromatography, dried, acetylated according to publishedprocedures and quantitated using radioimmunoassay procedures. Iodinatedligands of derivatized cyclic AMP and cyclic GMP are incubated withstandards or unknowns in the presence of specific antisera andappropriate buffers.

Verification of the cyclic nucleotide content may be obtained bydetermining the turnover or accumulation of cyclic nucleotides in intactcells. To measure intact cell cAMP, ³H-adenine pre-labeling is usedaccording to published procedures (Whalin, M. E., Garrett Jr., R. L.,Thompson, W. J., and Strada, S. J. “Correlation of cell-free braincyclic nucleotide phosphodiesterase activities to cyclic AMP decay inintact brain slices”, Sec. Mess. and Phos. Protein Research, 12:311-325,1989, which is incorporated herein by reference). The procedure measuresflux of labeled ATP to cyclic AMP and can be used to estimate intactcell adenylate cyclase or cyclic nucleotide phosphodiesterase activitiesdepending upon the specific protocol. Cyclic GMP accumulation was toolow to be studied with intact cell pre-labeling according to publishedprocedures (Reynolds, P. E., S. J. Strada and W. J. Thompson, “CyclicGMP Accumulation In Pulmonary Microvascular Endothelial Cells MeasuredBy Intact Cell Prelabeling,” Life Sci., 60:909-918, 1997, which isincorporated herein by reference).

The PDE inhibitory activity effect of a compound can also be determinedfrom a tissue sample. Tissue biopsies from humans or tissues fromanesthesized animals are collected from subjects exposed to the testcompound. Briefly, a sample of tissue is homogenized in 500 μl of 6%TCA. A known amount of the homogenate is removed for protein analysis.The remaining homogenate is allowed to sit on ice for 20 minutes toallow for the protein to precipitate. Next, the homogenate iscentrifuged for 30 minutes at 15,000 g at 4° C. The supernatant isrecovered, and the pellet recovered. The supernatant is washed fourtimes with five volumes of water saturated diethyl ether. The upperether layer is discarded between each wash. The aqueous ether extract isdried in a speed vac. Once dried, the sample can be frozen for futureuse, or used immediately. The dried extract is dissolved in 500 μl ofassay buffer. The amount of cGMP-specific inhibition is determined byassaying for the amount of cyclic nucleotides using RIA procedures asdescribed above.

The amount of inhibition is determined by comparing the activity of thenovel DE (or PDE2) in the presence and absence of the compound.Inhibition of the novel PDE activity (or PDE2) is indicative that thecompound is useful for treating neoplasia. Significant inhibitoryactivity greater than that of the benchmark, exisulind, preferablygreater than 50% at a concentration of 10 μM or below, is indicativethat a compound should be further evaluated for antineoplasticproperties. Preferably, the IC₅₀ value for the novel PDE inhibitionshould be less than 50 μM for the compound to be further considered forpotential use.

D. Determining Whether A Compound Reduces Tumor Cell Growth

In an alternate embodiment, the method of the present invention involvesfurther determining whether the compound reduces the growth of tumorcells. Various cell lines can be used in the sample depending on thetissue to be tested. For example, these cell lines include:SW-480—colonic adenocarcinoma; HT-29—colonic adenocarcinoma, A-427—lungadenocarcinoma carcinoma; MCF-7—breast adenocarcinoma; andUACC-375—melanoma line; and DU145—prostrate carcinoma. Cytotoxicity dataobtained using these cell lines are indicative of an inhibitory effecton neoplastic lesions. These cell lines are well characterized, and areused by the United States National Cancer Institute in their screeningprogram for new anti-cancer drugs.

A compound's ability to inhibit tumor cell growth can be measured usingthe HT-29 human colon carcinoma cell line obtained from ATCC. HT-29cells have previously been characterized as a relevant colon tumor cellculture model (Fogh, J., and Trempe, G. In: Human Tumor Cells in Vitro,J. Fogh (eds.), Plenum Press, New York, pp. 115-159, 1975). HT-29 cellsare maintained in RPMI media supplemented with 5% fetal bovine calfserum (Gemini Bioproducts, Inc., Carlsbad, Calif.) and 2 mm glutamine,and 1% antibiotic-antimycotic in a humidified atmosphere of 95% air and5% CO₂ at 37° C. Briefly, HT-29 cells are plated at a density of 500cells/well in 96 well microtiter plates and incubated for 24 hours at37° C. prior to the addition of compound. Each determination of cellnumber involved six replicates. After six days in culture, the cells arefixed by the addition of cold trichloroacetic acid to a finalconcentration of 10% and protein levels are measured using thesulforhodamine B (SRB) colorimetric protein stain assay as previouslydescribed by Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon,J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M.R., “New Colorimetric Assay For Anticancer-Drug Screening,” J. Natl.Cancer Inst. 82: 1107-1112, 1990, which is incorporated herein byreference.

In addition to the SRB assay, a number of other methods are available tomeasure growth inhibition and could be substituted for the SRB assay.These methods include counting viable cells following trypan bluestaining, labeling cells capable of DNA synthesis with BrdU orradiolabeled thymidine, neutral red staining of viable cells, or MTTstaining of viable cells.

Significant tumor cell growth inhibition greater than about 50% at adose of 100 μM or below is further indicative that the compound isuseful for treating neoplastic lesions. Preferably, an IC₅₀ value isdetermined and used for comparative purposes. This value is theconcentration of drug needed to inhibit tumor cell growth by 50%relative to the control. Preferably, the IC₅₀ value should be less than100 μM for the compound to be considered further for potential use fortreating neoplastic lesions.

E. Determining Whether A Compound Induces Apoptosis

In a second alternate embodiment, the screening method of the presentinvention further involves determining whether the compound inducesapoptosis in cultures of tumor cells.

Two distinct forms of cell death may be described by morphological andbiochemical criteria: necrosis and apoptosis. Necrosis is accompanied byincreased permeability of the plasma membrane; the cells swell and theplasma membrane ruptures within minutes. Apoptosis is characterized bymembrane blebbing, condensation of cytoplasm and the activation ofendogenous endonucleases.

Apoptosis occurs naturally during normal tissue turnover and duringembryonic development of organs and limbs. Apoptosis also is induced bycytotoxic T-lymphocytes and natural killer cells, by ionizing radiationand by certain chemotherapeutic drugs. Inappropriate regulation ofapoptosis is thought to play an important role in many pathologicalconditions including cancer, AIDS, or Alzheimer's disease, etc.Compounds can be screened for induction of apoptosis using cultures oftumor cells maintained under conditions as described above. Treatment ofcells with test compounds involves either pre- or post-confluentcultures and treatment for two to seven days at various concentrations.Apoptotic cells are measured in both the attached and “floating”compartments of the cultures. Both compartments are collected byremoving the supernatant, trypsinizing the attached cells, and combiningboth preparations following a centrifugation wash step (10 minutes, 2000rpm) The protocol for treating tumor cell cultures with sulindac andrelated compounds to obtain a significant amount of apoptosis has beendescribed in the literature. (See, Piazza, G. A., et al., CancerResearch, 55:3110-16, 1995, which is incorporated herein by reference).The novel features include collecting both floating and attached cells,identification of the optimal treatment times and dose range forobserving apoptosis, and identification of optimal cell cultureconditions.

Following treatment with a compound, cultures can be assayed forapoptosis and necrosis by florescent microscopy following labeling withacridine orange and ethidium bromide. The method for measuring apoptoticcell number has previously been described by Duke & Cohen,“Morphological And Biochemical Assays Of Apoptosis,” Current ProtocolsIn Immunology, Coligan et al., eds., 3.17.1-3.17.16 (1992, which isincorporated herein by reference).

For example, floating and attached cells can be collected bytrypsinization and washed three times in PBS. Aliquots of cells can becentrifuged. The pellet can then be re-suspended in media and a dyemixture containing acridine orange and ethidium bromide prepared in PBSand mixed gently. The mixture can then be placed on a microscope slideand examined for morphological features of apoptosis.

Apoptosis can also be quantified by measuring an increase in DNAfragmentation in cells that have been treated with test compounds.Commercial photometric EIA for the quantitative, in vitro determinationof cytoplasmic histoneassociated-DNA-fragments (mono- andoligonucleosomes) are available (Cell Death Detection ELISA^(okys), Cat.No. 1,774,425, Boehringer Mannheim). The Boehringer Mannheim assay isbased on a sandwich-enzyme-immunoassay principle using mouse monoclonalantibodies directed against DNA and histones, respectively. This allowsthe specific determination of mono- and oligonucleosomes in thecytoplasmatic fraction of cell lysates.

According to the vendor, apoptosis is measured in the following fashion.The sample (cell-lysate) is placed into a streptavidin-coated microtiterplate (“MTP”). Subsequently, a mixture of anti-histone-biotin andanti-DNA peroxidase conjugate are added and incubated for two hours.During the incubation period, the anti-histone antibody binds to thehistone-component of the nucleosomes and simultaneously fixes theimmunocomplex to the streptavidin-coated MTP via its biotinylation.Additionally, the anti-DNA peroxidase antibody reacts with the DNAcomponent of the nucleosomes. After removal of unbound antibodies by awashing step, the amount of nucleosomes is quantified by the peroxidaseretained in the immunocomplex.

Peroxidase is determined photometrically with ABTS7 (2,2′-Azido-[3ethylbenzthiazolin-sulfonate]) as substrate.

For example, SW-480 colon adenocarcinoma cells are plated in a 96-wellMTP at a density of 10,000 cells per well. Cells are then treated withtest compound, and allowed to incubate for 48 hours at 37° C. After theincubation, the MTP is centrifuged, and the supernatant is removed. Thecell pellet in each well is then resuspended in lysis buffer for 30minutes. The lysates are then centrifuged and aliquots of thesupernatant (i.e., the cytoplasmic fraction) are transferred into astreptavidin-coated MTP. Care is taken not to shake the lysed pellets(i.e. cell nucleii containing high molecular weight, unfragmented DNA)in the MTP. Samples are then analyzed.

Fold stimulation (FS=OD_(max)/OD_(veh)), an indicator of apoptoticresponse, is determined for each compound tested at a givenconcentration. EC₅₀ values may also be determined by evaluating a seriesof concentrations of the test compound.

Statistically significant increases in apoptosis (i.e., greater than 2fold stimulation at a concentration of 100 μM) are further indicativethat the compound is useful for treating neoplastic lesions. Preferably,the EC₅₀ value for apoptotic activity should be less than 100 μM for thecompound to be further considered for potential use for treatingneoplastic lesions. EC₅₀ is herein defined as the concentration thatcauses 50% induction of apoptosis relative to vehicle treatment.

F. Mammary Gland Organ Culture Model Tests

Test compounds identified by the above methods can be tested forantineoplastic activity by their ability to inhibit the incidence ofpre-neoplastic lesions in a mammary gland organ culture system. Thismouse mammary gland organ culture technique has been successfully usedby other investigators to study the effects of known antineoplasticagents such as certain NSAIDs, retinoids, tamoxifen, selenium, andcertain natural products, and is useful for validation of the screeningmethod of the present invention.

For example, female BALB/c mice can be treated with a combination ofestradiol and progesterone daily, in order to prime the glands to beresponsive to hormones in vitro. The animals are sacrificed, andthoracic mammary glands are excised aseptically and incubated for tendays in growth media supplemented with insulin, prolactin,hydrocortisone, and aldosterone. DMBA (7,12 dimethylbenz(a)anthracene)is added to medium to induce the formation of premalignant lesions.Fully developed glands are then deprived of prolactin, hydrocortisone,and aldosterone, resulting in the regression of the glands but not thepre-malignant lesions.

The test compound is dissolved in DMSO and added to the culture mediafor the duration of the culture period. At the end of the cultureperiod, the glands are fixed in 10% formalin, stained with alum carmine,and mounted on glass slides. The incidence of forming mammary lesions isthe ratio of the glands with mammary lesions to glands without lesions.The incidence of mammary lesions in test compound treated glands iscompared with that of the untreated glands.

The extent of the area occupied by the mammary lesions can bequantitated by projecting an image of the gland onto a digitation pad.The area covered by the gland is traced on the pad and considered as100% of the area. The space covered by each of the non-regressedstructures is also outlined on the digitization pad and quantitated bythe computer.

EXPERIMENTAL RESULTS

A number of compounds were examined in the various protocols andscreened for potential use in treating neoplasia. The results of thesetests are reported below. The test compounds are hereinafter designatedby a letter code that corresponds to the following:

A—rac-threo-(E)-1-(N,N′-diethylaminoethanethio)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;

B—(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-acetic acid;

C—(Z)-5-Fluoro-2-methyl-1-(p-chlorobenzylidene)-3-acetic acid;

D—rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-N-acetylcysteine;

E—(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetamide,N-benzyl;

F—(Z)-5-Fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetamide,N,N′-dicyclohexyl;

G—ribo-(E)-1-Triazolo-[2′,3′:1″,3″]-1(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;and

H—rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-glutathione).

EXAMPLE 1 COX Inhibition Assay

Reference compounds and test compounds were analyzed for their COXinhibitory activity in accordance with the protocol for the COX assay,supra. FIG. 4 hows the effect of various concentrations of eithersulindac sulfide or exisulind on urified cyclooxygenase (Type 1)activity. Cyclooxygenase activity was determined using purifiedcyclooxygenase from ram seminal vesicles as described previously(Mitchell et al, supra). The IC₅₀ value for sulindac sulfide wascalculated to be approximately 1.76 μM, while that for exisulind wasgreater than 10,000 μM. These data show that sulindac sulfide, but notexisulind, is a COX-I inhibitor. Similar data were obtained for theCOX-2 isoenzyme (Thompson, et al., Journal of the National CancerInstitute, 87: 1259-1260, 1995).

FIG. 5 shows the effect of test compounds B and E on COX inhibition. COXactivity was determined as for the compounds shown in FIG. 4. The datashow that neither test compound B and E significantly inhibit COX-I.

TABLE 2 Cyclooxygenase inhibitory activity for a series of compoundsReference compounds % Inhibition at 100 μM Indomethacin 95 MY5445 94Sulindac sulfide 97 Exisulind <25 Test compounds % Inhibition at 100 μMA <25 B <25 C 87 D <25 E <25

In accordance with the protocol, supra, compounds A through E wereevaluated for COX inhibitory activity as reported in Table 2 above.Compound C was found to inhibit COX greater than 25% at a 100 μM dose,and therefore, would not be selected for further screening.

EXAMPLE 2 cGMP PDE Inhibition Assay

Reference compounds and test compounds were analyzed for their cGMP PDEinhibitory activity in accordance with the protocol for the assaydescribed supra. FIG. 6 shows the effect of various concentrations ofsulindac sulfide and exisulind on either PDE4 or cGMP PDE activitypurified from human colon HT-29 cultured tumor cells, as describedpreviously (W. J. Thompson et al., supra). The IC₅₀ value of sulindacsulfide for inhibition of PDE4 was 41 μM, and for inhibition of cGMP PDEwas 17 μM. The IC₅₀ value of exisulind for inhibition of PDE4 was 181μM, and for inhibition of cGMP PDE was 56 μM. These data show that bothsulindac sulfide and exisulind inhibit phosphodiesterase activity. Bothcompounds show selectivity for the cGMP PDE isoenzyme forms over PDE4isoforms.

FIG. 7 shows the effects of sulindac sulfide on either cGMP or cAMPproduction as determined in cultured HT-29 cells in accordance with theassay described, supra. HT-29 cells were treated with sulindac sulfidefor 30 minutes and cGMP or cAMP was measured by conventionalradioimmunoassay method. As indicated, sulindac sulfide increased thelevels of cGMP by greater than 50% with an EC₅₀ value of 7.3 μM (FIG.7A). Levels of cAMP were unaffected by treatment, although a known PDE4inhibitor, rolipram, increased cAMP (FIG. 7B). The data demonstrate thepharmacological significance of inhibiting cGMP PDE, relative to PDE4.

FIG. 8 shows the effect of the indicated dose of test compound B oneither cGMP PDE or PDE4 isozymes of phosphodiesterase. The calculatedIC₅₀ value was 18 μM for cGMP PDE and was 58 μM for PDE4.

FIG. 9 shows the effect of the indicated dose of test compound E oneither PDE4 or cGMP PDE. The calculated IC₅₀ value was 0.08 μM for cGMPPDE and greater than 25 μM for PDE4.

TABLE 3 cGMP PDE inhibitory activity among a series of compoundsReference compounds % Inhibition at 10 μM Indomethacin 34 MY5445 86Sulindac sulfide 97 Exisulind 39 Test compounds % Inhibition at 10 μM A<25 B <25 C <25 D 36 E 75

The above compounds in Table 3 were evaluated for PDE inhibitoryactivity, as described in the protocol supra. Of the compounds that didnot inhibit COX, only compound E was found to cause greater than 50%inhibition at 10 μM. As noted in FIG. 8, compound B showed inhibition ofgreater than 50% at a dose of 20 μM. Therefore, depending on the dosagelevel used in a single dose test, some compounds may be screened outthat otherwise may be active at slightly higher dosages. The dosage usedis subjective and may be lowered after active compounds are found atcertain levels to identify even more potent compounds.

EXAMPLE 3 Apoptosis Assay

Reference compounds and test compounds were analyzed for their novel PDEinhibitory activity in accordance with the protocols for the assay,supra. In accordance with those protocols, FIG. 10 shows the effects ofsulindac sulfide and exisulind on apoptotic and necrotic cell death.HT-29 cells were treated for six days with the indicated dose of eithersulindac sulfide or exisulind. Apoptotic and necrotic cell death wasdetermined previously (Duke and Cohen, In: Current Protocols inImmunology, 3.17.1 -3.17.16, New York, John Wiley and Sons, 1992). Thedata show that both sulindac sulfide and exisulind are capable ofcausing apoptotic cell death without inducing necrosis. All data werecollected from the same experiment.

FIG. 11 shows the effect of sulindac sulfide and exisulind on tumorgrowth inhibition and apoptosis induction as determined by DNAfragmentation. Top FIG. (11A); growth inhibition (open symbols, leftaxis) and DNA fragmentation (closed symbols, right axis) by exisulind.Bottom FIG. (11B); growth inhibition (open symbols) and DNAfragmentation (closed symbols) by sulindac sulfide. Growth inhibitionwas determined by the SRB assay after six days of treatment. DNAfragmentation was determined after 48 hours of treatment. All data werecollected from the same experiment.

FIG. 12 shows the apoptosis inducing properties of compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound E for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was 0.05 μM.

FIG. 13 shows the apoptosis inducing properties of compound B. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound B for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was approximately 175 μM.

TABLE 4 Apoptosis-inducing activity among a series of compoundsReference compounds Fold induction at 100 μM Indomethacin <2.0 MY54454.7 Sulindac sulfide 7.9 Exisulind <2.0 E4021 <2.0 Zaprinast <2.0Sildenafil <2.0 EHNA <2.0 Test compounds Fold induction at 100 μM A <2.0B 3.4 C 5.6 D <2.0 E 4.6

In accordance with the fold induction protocol, supra, the compounds Athrough E were tested for apoptosis inducing activity, as reported inTable 4 above. Compounds B, C and E showed significant apoptoticinducing activity, greater than 2.0 fold, at a dosage of 100 μM. Ofthese three compounds, at this dosage only B and E did not inhibit COXbut did inhibit cGMP-specific PDE.

The apoptosis inducing activity for a series of phosphodiesteraseinhibitors was determined. The data are presented in Table 5 below.HT-29 cell were treated for 6 days with various inhibitors ofphosphodiesterase. Apoptosis and necrosis were determinedmorphologically after acridine orange and ethidium bromide labeling inaccordance with the assay described, supra. The data show that the novelcGM-Pspecific PDE is useful for screening compounds that induceapoptosis of HT-29 cells.

TABLE 5 Apoptosis-Induction Data for PDE Inhibitors Inhibitor ReportedSelectivity % Apoptosis % Necrosis Vehicle 8 6 8-methoxy-IBMX PDE1 2 1Milrinone PDE3 18 0 RO-20-1724 PDE4 11 2 MY5445 PDE5 80 5 IBMXNon-selective 4 13

EXAMPLE 4 Growth Inhibition Assay

Reference compounds and test compounds were analyzed for their PDE5inhibitory activity in accordance with the protocol for the assay supra.FIG. 14 shows the inhibitory effect of various concentrations ofsulindac sulfide and exisulind on the growth of HT-29 cells. HT-29 cellswere treated for six days with various doses of exisulind (triangles) orsulindac sulfide (squares) as indicated. Cell number was measured by asulforhodamine assay as previously described (Piazza et al., CancerResearch, 55: 3110-3116, 1995). The IC₅₀ value for sulindac sulfide wasapproximately 45 μM and 200 μM for the exisulind. The data show thatboth sulindac sulfide and exisulind are capable of inhibiting tumor cellgrowth.

FIG. 15 shows the growth inhibitory and apoptosis-inducing activity ofsulindac sulfide. A time course experiment is shown involving HT-29cells treated with either vehicle, 0.1% DMSO (open symbols) or sulindacsulfide, 120 μM (closed symbols). Growth inhibition (15A top) wasmeasured by counting viable cells after trypan blue staining. Apoptosis(15B bottom) was measured by morphological determination followingstaining with acridine orange and ethidium bromide as describedpreviously (Duke and Cohen, in: Current Protocols in Immunology,3.17.1-3.17.16, New York, John Wiley and Sons, 1992). The datademonstrate that sulindac sulfide is capable of inhibiting tumor cellgrowth, and that the effect is accompanied by an increase in apoptosis.All data were collected from the same experiment.

FIG. 16 shows the growth inhibitory activity of test compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound E for six days and cell number was determined by the SRBassay. The calculated IC₅₀ value was 0.04 μM.

TABLE 6 Growth-inhibitory activity among a series of compounds Referencecompounds % Inhibition at 100 μM Indomethacin 75 MY5445 88 Sulindacsulfide 88 Exisulind <50 E4021 <50 sildenafil <50 zaprinast <50 Testcompounds % Inhibition at 100 μM A 68 B 77 C 80 D 78 E 62

In accordance with the screening protocol of section supra, compounds Athrough E were tested for growth inhibitory activity, as reported inTable 6 above. All the test compounds showed activity exceeding a 100 μMsingle dose test.

The growth inhibitory activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 7 below. HT-29cells were treated for 6 days with various inhibitors ofphosphodiesterase. Cell growth was determined by the SRB assaydescribed, supra. The data below taken with those above show thatinhibitors of the novel PDE were effective for inhibiting tumor cellgrowth.

TABLE 7 Growth Inhibitory Data for PDE Inhibitors Growth inhibitionInhibitor Reported Selectivity (IC₅₀, μM) 8-methoxy-IBMX PDE1 >200 μMMilrinone PDE3 >200 μM RO-20-1724 PDE4 >200 μM MY5445 PDE5 5 μM IBMXNon-selective >100 μM Zaprinast PDE5 >100 μM Sildenafil PDE5 >100 μME4021 PDE5 >100 μM

To show the effectiveness of this screening method on various forms ofneoplasia, compounds were tested on numerous cell lines. The effects ofsulindac sulfide and exisulind on various cell lines were determined.The data are shown in Table 8 below. The IC₅₀ values were determined bythe SRB assay. The data show the broad effectiveness of these compoundson a broad range of neoplasias, with effectiveness at comparable doserange. Therefore, compounds identified and selected by this inventionshould be useful for treating multiple forms of neoplasia.

TABLE 8 Growth Inhibitory Data of Various Cell Lines IC₅₀(μM) Com- CellType/ Sulindac pound Tissue specificity sulfide Exisulind E* HT-29,Colon 60 120 0.10 HCT116, Colon 45 90 MCF7/S, Breast 30 90 UACC375,Melanoma 50 100 A-427, Lung 90 130 Bronchial Epithelial Cells 30 90 NRK,Kidney (non ras-transformed) 50 180 KNRK, Kidney (ras transformed) 60240 Human Prostate Carcinoma PC3 82 0.90 Colo 205 1.62 DU-145 0.10HCT-15 0.60 MDA-MB-231 0.08 MDA-MB-435 0.04 *Determined by neutral redassay as described by Schmid et al., in Proc. AACR Vol 39, p. 195(1998).

EXAMPLE 5 Activity in Mammary Gland Organ Culture Model

FIG. 17 shows the inhibition of premalignant lesions in mammary glandorgan culture by sulindac metabolites. Mammary gland organ cultureexperiment were performed as previously described (Mehta and Moon,Cancer Research, 46:5832-5835, 1986). The results demonstrate thatsulindac and exisulind effectively inhibit the formation of premalignantlesions, while sulindac sulfide was inactive. The data support thehypothesis that cyclooxygenase inhibition is not necessary for theanti-neoplastic properties of desired compounds.

ANALYSIS

To select compounds for treating neoplasia, this invention provides arationale for comparing experimental data of test compounds from severalprotocols. Within the framework of this invention, test compounds can beranked according to their potential use for treating neoplasia inhumans. Those compounds having desirable effects may be selected foradditional testing and subsequent human use.

Qualitative data of various test compounds and the several protocols areshown in Table 9 below. The data show that exisulind, compound B andcompound E exhibit the appropriate activity to pass the screen of fourassays: lack of COX inhibition, and presence of effective cGMP-specificPDE inhibition, growth inhibition and apoptosis induction. The activityof these compounds in the mammary gland organ culture validates theeffectiveness of this invention. The qualitative valuations of thescreening protocols rank compound E best, then compound B and thenexisulind.

TABLE 9 Activity Profile of Various Compounds Mammary Gland COX PDEGrowth Organ Compound Inhibition Inhibition Inhibition Apoptosis CultureExisulind − ++ ++ ++ +++ Sulindac ++++ +++ +++ +++ − sulfide MY5445 +++++++ +++ +++ + A − − +++ ++ ++ B − +++ +++ +++ ++ D − − ++ − − E − ++++++++ ++++ ++++ F − − ++ + − G − − +++ ++ +++ H − − ++ − − Table 9 Code:Activity of compounds based on evaluating a series of experimentsinvolving tests for maximal activity and potency. −Not active +Slightlyactive ++Moderately active +++Strongly active ++++Highly active

Also disclosed is a novel assay for PKG activity, which is used in thescreening methods of this invention, but also has more generalusefulness in assaying for PKG activity for other purposes (e.g., forstudying the role of PKG in normal cellular function). For explanationpurposes, it is useful to describe the PKG assay first, beforedescribing how PKG activity can be useful in drug evaluation inascertaining whether a compound is potentially useful in the treatmentof neoplasia.

The Novel PKG Assay

The novel PKG assay of this invention involves binding to a solid phaseplural amino acid sequences, each of which contain at least the cGMPbinding domain and the phosphorylation site of phosphodiesterase type 5(“PDE5”). That sequence is known and described in the literature below.Preferably, the bound PDE5 sequence does not include the catalyticdomain of PDE5 as described below. One way to bind the PDE5 sequences toa solid phase is to express those sequences as a fusion protein of thePDE5 sequence and one member of an amino acid binding pair, andchemically link the other member of that amino acid binding pair to asolid phase (e.g., beads). One binding pair that can be used isglutathione S-transferase (“GST”) and glutathione (“GSH”), with the GSTbeing expressed as a fusion protein with the PDE5 sequence describedabove, and the GSH bound covalently to the solid phase. In this fashion,the PDE5 sequence/GST fusion protein can be bound to a solid phasesimply by passing a solution containing the fusion protein over thesolid phase, as described below.

RT-PCR method is used to obtain the cGB domain of PDE5 with forward andreverse primers designed from bovine PDE5 A cDNA sequence(McAllister-Lucas L. M. et al, J. Biol. Chem. 268, 22863-22873, 1993)and the selection among PDE 1-10 families. 5′-3′, Inc. kits for totalRNA followed by oligo (dT) column purification of mRNA are used withHT-29 cells. Forward primer (GAA-TTC-TGT-TAG-AAA-AGC-CAC-CAG-AGA-AAT-G,203-227) and reverse primer (CTC-GAG-CTCTCT-TGT-TTC-TTC-CTC-TGC-TG,1664-1686) are used to synthesize the 1484 bp fragment coding for thephosphorylation site and both low and high affinity cGMP binding sitesof human PDESA (203-1686 bp, cGB-PDE5). The synthesized cGBPDE5nucleotide fragment codes for 494 amino acids with 97% similarity tobovine PDE5 A. It is then cloned into pGEX-5 X-3glutathione-S-transferase (GST) fusion vector (Pharmacia Biotech )withtac promoter, and EcoRI and XhoI cut sites. The fusion vector is thentransfected into E. Coli BL21 (DE3) bacteria (Invitrogen). Thetransfected BL21 bacteria is grown to log phase and then IPTG is addedas an inducer. The induction is carried at 20° C. for 24 hrs. Thebacteria are harvested and lysated. The soluble cell lysate is incubatedwith GSH conjugated Sepharose 4 B (GSHSepharose 4 B). The GST-cGB-PDE5fusion protein can bind to the GSH-Sepharose beads and the otherproteins are washed off from beads with excessive cold PBS.

The expressed GST-cGB-PDE5 fusion protein is displayed on 7.5% SDS-PAGEgel as a 85 Kd protein. It is characterized by its cGMP binding andphosphorylation by protein kinases G and A. It displays two cGMP bindingsites and the Kd is 1.6±0.2 μM, which is close to K_(d)=1.3 μM of thenative bovine PDE5. The GST-cGB-PDE5 on GSH conjugated sepharose beadscan be phosphorylated in vitro by cGMP-dependent protein kinase andcAMP-dependent protein kinase A. The K_(m) of GST-cGB-PDE5Phosphorylation by PKG is 2.7 μM and Vmax is 2.8 μM, while the K_(m) ofBPDEtide phosphorylation is 68 μM. The phosphorylation by PKG shows onemolecular phosphate incorporated into one GST-cGB-PDE5 Protein ratio.

To assay a liquid sample believed to contain PKG using the PDE5-boundsolid phase described above, the sample and the solid phase are mixedwith phosphorylation buffer containing ³²P-γ-ATP. The solution isincubated for 30 minutes at 30° C. to allow for phosphorylation of thePDE5 sequence by PKG to occur, if PKG is present. The solid phase isthen separated from solution (e.g., by centrifugation or filtration) andwashed with phosphate-buffered saline (“PBS”) to remove any remainingsolution and to remove any unreacted ³²P-γ-ATP.

The solid phase can then be tested directly (e.g., by liquidscintillation counter) to ascertain whether ³²P is incorporated. If itdoes, that indicates that the sample contained PKG since PKGphosphorylates PDE5. If the DE5 is bound via fusion protein, asdescribed above, the PDE5-containing fusion protein can be eluted fromthe solid phase with SDS buffer, and the eluent can be assayed for³²Pincorporation. This is particularly advantageous if there is thepossibility that other proteins are present, since the eluent can beprocessed (e.g., by gel separation) to separate various proteins fromeach other so that the fusion protein fraction can be assayed for³²Pincorporation. The phosphorylated fusion protein can be eluted from thesolid phase with SDS buffer and further resolved by electrophoresis. Ifgel separation is performed, the proteins can be stained to see theposition(s) of the protein, and ³²P phosphorylation of the PDE5 Portionof the fusion protein by PKG can be measured by X-ray film exposure tothe gel. If ³²P is made visible on X-ray film, that indicates that PKGwas present in the original sample contained PKG, which phosphorylatedthe PDE5 Portion of the fusion protein eluted from the solid phase.

Preferably in the assay, one should add to the assay buffer an excess(e.g., 100 fold) of protein kinase inhibitor (“PKI”) which specificallyand potently inhibits protein kinase A (“PKA”) without inhibiting PKG.Inhibiting PKA is desirable since it may contribute to thephosphorylation of the PKG substrate (e.g., PDE5). By adding PKI, anycontribution to phosphorylation by PKA will be eliminated, and anyphosphorylation detected is highly likely to be due to PKG alone.

A kit can be made for the assay of this invention, which kit containsthe following pre-packaged reagents in separate containers:

1. Cell lysis buffer: 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA,1 mM Na₃VO_(4, 1) mM NaF, 500 μM IBMX, proteinase inhibitors.

2. Protein kinase G solid phase substrate: recombinant GST-cGB-PDE5bound Sepharose 4 B (50% slurry).

3. 2× Phosphorylation buffer: ³²P-γ-ATP (3000 mCi/mmol, 5˜10 μCi/assay),10 mM KH₂PO_(4, 10) mM K₂HPO_(4, 200) μM ATP, 5 mM MgCl₂.

4. PKA Protein Kinase I Inhibitor

Disposable containers and the like in which to perform the abovereactions can so be provided in the kit.

From the above, one skilled in the analytical arts will readily envisionvarious ways to adapt the assay formats described to still otherformats. In short, using at least a portion of PDE5 (or any otherprotein that can be selectively phosphorylated by PKG), the presence andrelative amount (as compared to a control) of PKG can be ascertained byevaluating phosphorylation of the phosphorylatable protein, using alabeled phosphorylation agent.

SAANDs Increase PKG Activity In NeoDlastic Cells

Using the PKG assay described above, the following experiments wereperformed to establish that SAANDs increase PKG activity due either toincrease in PKG expression or an increase in cGMP levels (or both) inneoplastic cells treated with a SAAND.

Test Procedures

Two different types of PDE inhibitors were evaluated for their effectson PKG in neoplastic cells. A SAAND, exisulind, was evaluated since itis anti-neoplastic. Also, a non-SAAND classic PDE5 inhibitor, E4021, wasevaluated to ascertain whether PKG elevation was simply due to classicPDE5 inhibition, or whether PKG elevation was involved in thepro-apoptotic effect of SAANDs inhibition of PDE5 and the novel PDEdisclosed in United States Patent Application No. 09/173,375 to Liu etal filed Oct. 15, 1998.

To test the effect of cGMP-specific PDE inhibition on neoplasiacontaining the APC mutation, SW480 colon cancer cells were employed. SW480 is known to contain the APC mutation. About 5 million SW480 cells inRPMI 5% serum are added to each of 8 dishes:

2-10 cm dishes—30 μL DMSO vehicle control (without drug),

3-10 cm dishes—200 μM, 400 μM, 600 μM exisulind in DMSO, and

3-10 cm dishes—E4021;0.1 μM, 1 μM and 10 μM in DMSO.

The dishes are incubated for 48 hrs at 37° C. in 5% CO₂ incubator.

The liquid media are aspirated from the dishes (the cells will attachthemselves to the dishes). The attached cells are washed in each dishwith cold PBS, and 200 μL cell lysis buffer (i.e., 50 mM Tris-HCl, 1%NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO_(4, 1) mM NaF, 500 μM IBMXwith proteinase inhibitors) is added to each dish. Immediately after thecell lysis buffer is added, the lysed cells are collected by scrapingthe cells off each dish. The cell lysate from each dish is transferredto a microfuge tube, and the microfuge tubes are incubated at 4° C. for15 minutes while gently agitating the microfuge tubes to allow the cellsto lyse completely. After lysis is complete, the microfuge tubes arecentrifuged full speed (14,000 r.p.m.) for 15 minutes. The supernatantfrom each microfuge tube is transferred to a fresh microfuge tube.

A protein assay is then performed on the contents of each microfuge tubebecause the amount of total protein will be greater in the control thanin the drugtreated samples, if the drug inhibits cell growth. Obviously,if the drug does work, the total protein in the drug-treated samplesshould be virtually the same as control. In the above situation, thecontrol and the E-4021 microfuge tubes needed dilution to normalize themto the high-dose exisulind-treated samples (the lower dose groups ofexisulind had to be normalized to the highest dose exisulind sample).Thus, after the protein assays are performed, the total proteinconcentration of the various samples must be normalized (e.g., bydilution).

For each drug concentration and control, two PKG assays are performed,one with added cGMP, and one without added cGMP, as described in detailbelow. The reason for performing these two different PKG assays is thatcGMP specifically activates PKG. When PKG activity is assayed using thenovel PKG assay of this invention, one cannot ascertain whether anyincrease the PKG activity is due to increased cGMP in the cells (thatmay be caused by cGMP-specific PDE inhibition) or whether the PKGactivity level is due to an increased expression of PKG protein. Bydetermining PKG activity in the same sample both with and without addedcGMP, one can ascertain whether the PKG activity increase, if any, isdue to increased PKG expression. Thus, if an anti-neoplastic drugelevates PKG activity relative to control, one can establish if thedrug-induced increase is due to increased PKG protein expression (asopposed to activation) in the drug-treated sample if (1) thedrug-treated sample with extra cGMP exhibits greater PKG activitycompared to the control sample with extra cGMP, and (2) the drug-treatedsample without extra cGMP exhibits greater PKG activity relative tocontrol.

After, parallel samples with and without added cGMP are prepared, 50 μLof each cell lysate is added to 20 μL of the PDE5/GST solid phasesubstrate slurry described above. For each control or drug cell lysatesample to be evaluated, the reaction is started by addingphosphorylation buffer containing 10 μCi ³²P-γ-ATP solution (200 μM ATP,4.5 mM MgCl; 5 mM KH₂PO₄;5 mM K₂HP0₄;) to each mixture. The resultantmixtures are incubated at 30° C. for 30 minutes. The mixtures are thencentrifuged to separate the solid phase, and the supernatant isdiscarded. The solid phase in each tube is washed with 700 μL cold PBS.To the solid phase, Laemmli sample buffer (Bio-Rad) (30 μL) is added.The mixtures are boiled for 5 minutes, and loaded onto 7.5% SDS-PAGE.The gel is run at 150 V for one hour. The bands obtained are stainedwith commassie blue to visualize the 85 Kd GST-PDE5 fusion proteinbands, if present. The gel is dried, and the gel is laid on x-ray filmwhich, if the PDE5 is phosphorylated, the film will show a correspondingdarkened band. The darkness of each band relates to the degree ofphosphorylation.

As shown in FIGS. 18A and 18B, the SAAND exisulind causes PKG activityto increase in a dose-dependent manner in both the samples with addedcGMP and without added cGMP relative to the control samples with andwithout extra cGMP. This is evidenced by the darker appearances of the85 Kd bands in each of the drug-treated samples. In addition, the SW480samples treated with exisulind show a greater PKG phosphorylationactivity with added cGMP in the assay relative to the samples treatedwith exisulind alone (i.e. no added cGMP). Thus, the increase in PKGactivity in the drug-treated samples is not due only to the activationof PKG by the increase in cellular cGMP when the SAAND inhibitscGMP-specific PDE, the increase in PKG activity in neoplasia harboringthe APC mutation is due to increased PKG expression as well.

Also the fact that the E4021-treated SW480 samples do not exhibit PKGactivation relative to control (see FIGS. 18A and 18B) shows that theincreased PKG activation caused by SAANDs in neoplasia containing theAPC mutation is not simply due to inhibition of classic PDE5.

As an analytic technique for evaluating PKG activation, instead of x-rayfilm exposure as described above, the 85 Kd band from the SDS page canbe evaluated for the degree of phosphorylation by cutting the band fromthe gel, and any ³²P incorporated in the removed band can be counted byscintillation (beta) counter in the ³²P window.

To test the effect of cGMP-specific PDE inhibition on neoplasiacontaining the β-catenin mutation, HCT116 colon cancer cells wereemployed. HCT116 is known to contain the β-catenin mutation, but isknown not to contain the APC mutation.

The same procedure is used to grow the HCT116 cells as is used in theSW480 procedure described above. In this experiment, only exisulind andcontrols were used. The exisulind-treated cells yielded PKG that wasphosphorylated to a greater extent than the corresponding controls,indicating that PKG activation occurred in the drug-treated cells thatis independent of the APC mutation.

Thus, for the purposes of the present invention, we refer to “reducingβ-catenin” in the claims to refer to wild type and/or mutant forms ofthat protein.

Confirmation of Increased PKG Expression and Decreased β-Catenin In SW480 By Western Blot

As demonstrated above, SAANDs cause an increase in PKG expression and anincrease in cGMP level, both of which cause an increase in PKG activityin SAANDs-treated neoplastic cells. This increase in PKG proteinexpression was further verified by relatively quantitative western blot,as described below.

SW480 cells treated with exisulind as described previously are harvestedfrom the microfuge tubes by rinsing once with ice-cold PBS. The cellsare lysed by modified RIPA buffer for 15 minutes with agitation. Thecell lysate is spun down in a cold room. The supernatants aretransferred to fresh microcentrifuge tubes immediately after spinning.BioRad DC Protein Assay (Temecula, Calif.) is performed to determine theprotein concentrations in samples. The samples are normalized forprotein concentration, as described above.

50 μg of each sample is loaded to 10% SDS gel. SDS-PAGE is performed,and the proteins then are transferred to a nitrocellulose membrane. Theblotted nitrocellulose membrane are blocked in freshly prepared TBSTcontaining 5% nonfat dry milk for one hour at room temperature withconstant agitation.

A goat-anti-PKG primary antibody is diluted to the recommendedconcentration/dilution in fresh TBST/5% nonfat dry milk. Thenitrocellulose membrane is placed in the primary antibody solution andincubated one hour at room temperature with agitation. Thenitrocellulose membrane is washed three times for ten minutes each withTBST. The nitrocellulose membrane is incubated in a solution containinga secondary POD conjugated rabbit anti-goat antibody for 1 hour at roomtemperature with agitation. . The nitrocellulose membrane is washedthree times for ten minutes each time with TBST. The detection isperformed by using Boehringer Mannheim BM blue POD substrate.

As graphically illustrated in FIG. 19, exisulind causes the drop ofβ-catenin and the increase of PKG, which data were obtained by Westernblot. SW480 cells were treated with exisulind or vehicle (0.1% DMSO) for48 hours. 50 μg supernatant of each cell lysates were loaded to 10%SDS-gel and blotted to nitrocellulose membrane, and the membrane wasprobed with rabbit-anti-β-catenin and rabbit anti-PKG antibodies.

SAANDs Reduce β-Catenin Levels in Neoplastic Cells

This observation was made by culturing SW480 cells with either 200, 400or 600 μM exisulind or vehicle (0.1% DMSO). The cells are harvested 48hours post treatment and processed for immunoblotting. Immuno-reactiveprotein can be detected by Western blot. Western blot analysisdemonstrated that expression of β-catenin was reduced by 50% in theexisulind-treated cells as compared to control. These results indicatethat β-catenin is reduced by SAANDs treatment. Together with the resultsabove establishing PKG activity increases with such treatment and theresults below establishing that β-catenin is phosphorylated by PKG,these results indicate that the reduction of β-catenin in neoplasticcells is initiated by activation of PKG. Thus, using PKG activity inneoplasia as a screening tool to select compounds as anti-neoplastics isuseful.

The Phosphorylation of β-catenin By PKG

In vitro, PKG phosphorylates β-catenin. The experiment that establishedthis involves immunoprecipitating the β-catenin-containing complex fromSW480 cells (not treated with any drug) in the manner described belowunder “β-catenin immunoprecipitation” The immunoprecitated complex,while still trapped on the solid phase (i.e., beads) is mixed with³²P-γ-ATP and pure PKG (100 units). Corresponding controls with outadded PKG are prepared.

The protein is released from the solid phase by SDS buffer, and theproteincontaining mixture is run on a 7.5%SDS-page gel. The running ofthe mixture on the gel removes excess ³²P-γ-ATP from the mixture. Any³²P-γ-ATP detected in the 93 Kd β-catenin band, therefore, is due to thephosphorylation of the β-catenin. Any increase in ³²β-γ-ATP detected inthe 93 Kd β-catenin band treated with extra PKG relative to the controlwithout extra PKG, is due to the phosphorylation of the β-catenin in thetreated band by the extra PKG.

The results we obtained were that there was a noticeable increase inphosphorylation in the band treated with PKG as compared to the control,which exhibited minimal, virtually undetectable phosphorylation. Thisresult indicates that β-catenin can be phosphorylated by PKG.

The Phosphorylation of Mutant β-catenin By PKG

The same procedure described in the immediately preceding section wasperformed with HCT116 cells, which contain no APC mutation, but containa β-catenin mutation. The results of those experiments also indicatethat mutant β-catenin is phosphorylated by PKG.

Thus, for the purposes of the present invention, we refer to thephosphorylation of β-catenin in the claims to refer to thephosphorylation of wild type and/or mutant forms of that protein.

β-Catenin Precipitates With PKG

Supernatants of both SW480 and HCT116 cell lysates are prepared in thesame way described above in the Western Blot experiments. The celllysate are pre-cleared by adding 150 μl of protein A Sepharose beadslurry (50%) per 500 μg of cell lysate and incubating at 4° C. for 10minutes on a tube shaker. The protein A beads are removed bycentrifugation at 14,000×g at 4° C. for 10 minutes. The supernatant aretransferred to a fresh centrifuge tube. 10 μg of the rabbit polyclonalanti-β-catenin antibody (Upstate Biotechnology, Lake Placid, N.Y.) areadded to 500 μg of cell lysate. The cell lysate/antibody mixture isgently mixed for 2 hours at 4° C. on a tube shaker. The immunocomplex iscaptured by adding 150 μl protein A Sepharose bead slurry (75 μl packedbeads) and by gently rocking the mixture on a tube shaker for overnightat 4° C. The Sepharose beads are collected by pulse centrifugation (5seconds in the microcentrifuge at 14,000 rpm). The supernatant fractionis discarded, and the beads are washed 3 times with 800 μl ice-cold PBSbuffer. The Sepharose beads are resuspended in 150 μl 2×sample bufferand mixed gently. The Sepharose beads are boiled for 5 minutes todissociate the immunocomplexes from the beads. The beads are collectedby centrifugation and SDS-PAGE is performed on the supernatant.

A Western blot is run on the supernatant, and the membrane is thenprobed with an rabbit anti β-catenin antibody. Then the membrane iswashed 3 times for 10 minutes each with TBST to remove excess antiβ-catenin antibody. A goat, anti-rabbit antibody conjugated tohorseradish peroxidase is added, followed by 1 hour incubation at roomtemperature. When that is done, one can visualize the presence ofβ-catenin with an HRPO substrate. In this experiment, we could clearlyvisualize the presence of β-catenin.

To detect PKG on the same membrane, the anti-β-catenin antibodyconjugate is first stripped from the membrane with a 62 mM tris-HClbuffer (pH 7.6) with 2% SDS and 100 μM 2β-mercaptoethanol in 55° C.water bath for 0.5 hour. The stripped membrane is then blocked in TBSTwith 5% non-fat dried milk for one hour at room temperature whileagitating the membrane. The blocked, stripped membrane is then probedwith rabbit polyclonal anti-PKG antibody (Calbiochem, LaJolla, Calif.),that is detected with goat, anti-rabbit second antibody conjugated toHRPO. The presence of PKG on the blot membrane is visualized with anHRPO substrate. In this experiment, the PKG was, in fact, visualized.Given that the only proteins on the membrane are those thatimmunoprecipitated with β-catenin in the cell supernatants, this resultclearly establishes that PKG was physically linked to the proteincomplex containing the β-catenin in the cell supernatants.

The same Western blot membrane was also probed after stripping withantiGSK3-β antibody to ascertain whether it also co-precipitated withβ-catenin. In that experiment, we also detected GSK3-β on the membrane,indicating that the GSK3-β precipitated with the GSK3-β and PKG,suggesting that the three proteins may be part of the same complex.Since GSK3-β and β-catenin form part of the APC complex in normal cells,this that PKG may be part of the same complex, and may be involved inthe phosphorylation of β-catenin as part of that complex.

Anti-Neoplastic Pharmaceutical Compositions Containing cGMP PDEInhibitors

As explained above, exisulind is one compound that exhibits desirableantineoplastic properties. Its efficacy and use as an anti-neoplasticwas discovered before it was understood that the compound acted byinhibiting cGMP-specific PDE activity in neoplastic cells.

Among other things, the verification that the selection process of thisinvention could be used to select compounds for human treatment wasobtained in human clinical trials in patients with neoplasias. Byunderstanding after the fact that exisulind was anti-neoplastic (invitro), that it had the profile of a desirable compound meeting theselection criterion of this invention, the success of the compound intwo human clinical trials establishes that other compounds can beselected meeting the selection criterion of this invention.

As indicated above, a number of neoplasias harbor the APC mutation.Among other things, the verification of the selection process of thisinvention was established in human clinical trials in patients withneoplasia harboring the APC mutation.

The APC mutation was first discovered in patients with the hereditaryneoplasia, adenomatous polyposis coli (“APC”). The APC disease ischaracterized by the appearance in the teen years of hundreds tothousands of polyps in the colon, and the common therapy is surgicalremoval of the colon before the age of 20.

The first clinical trial involved patients with APC. using exisulind. Inthat study, each patient had already had his/her colon removed, exceptfor a small section of colon adjacent the rectum (where the smallintestine was attached) to preserve rectal finction. However, such apatient commonly forms polyps in the small remaining colonic section,which polyps require periodic removal (e.g., by electrocautery).

That trial where exisulind was selected was a prevention trial designedto evaluate the anti-neoplastic characteristics of the drug by comparingthe cumulative number of new polyps formed over twelve months by thedrug and placebo groups. Eligible patients were those who form between 9and 44 Polyps per year. Patients were fully ablated (had all polypsremoved) at the start of the study, at the end of 6 months and at theend of 12 months. The study enrolled thirty-four eligible patients.Based on the estimated mean number of polyps formed over a year in APCpatients who had historically produced 9 to 44 Polyps per year,exisulind was clinically and statistically significantly better thanplacebo in decreasing the rate of polyp formation. Based on the mediannumber of polyps produced in the first six months of the study, patientstreated with exisulind developed approximately one-third the number ofpolyps as patients treated with placebo (median values 9 Polyps/year and26 polyps/year, respectively; p=0.013). Based on the median number ofpolyps produced over the entire 12 months of the study, patients treatedwith exisulind produced approximately half the number of polyps aspatients treated with placebo (median values 18 Polyps/year and 38Polyps/year, respectively; p=0.020).

A separate clinical trial was also performed on male patients who hadprostate cancer, and as a result had their prostates removed. The studywas conducted in patients with detectable PSA (prostate specificantigen) levels that were rising following radical prostatectomy,indicating recurrence of prostate cancer.

96 Patients were enrolled in the prostate cancer evaluation: adouble-blind, placebo-controlled, multi-center trial involving exisulindadministered to the drug-receiving patients at 500 mg/day. As presentedbelow, the data show a statistically significant difference in PSAlevels between the exisulind-treated group and the placebo-treatedgroup. PSA levels in the exisulind-treated group were significantlyreduced as compared with the PSA levels of the placebo-treated group.Although a rising level of PSA is not itself a disease condition, it iswidely regarded in the medical community as a surrogate markerindicative of the presence of recurrence of prostate cancer in such men.

In addition to performing an evaluation based on the differences in meanPSA levels between the exisulind and placebo groups as a whole, theinterim analysis included subgroup analysis. The patients in the studywere classified into high, intermediate and low risk groups in terms oftheir risk of developing metastatic disease. This classification wasperformed using the methodology published in the Journal of the AmericanMedical Association (JAMA May 5, 1999, pp. 1591-97). To ascertain whichstudy patients fell into which risk group, medical histories weresupplied to a researcher who was blinded as to whether patients were ondrug or placebo; he assigned study patients to the appropriate riskgroups according to the above referenced published methodology. Thestatistical analysis revealed statistically significant differences inmean PSA levels between exisulind and placebo patients in both high andintermediate risk groups.

The data from the prostate study are as follows:

TABLE 10 Effect of Exisulind On Mean PSA Level In Men Post-ProstatectomyWith Rising PSA Group Placebo Exisulind “p” value Overall 4.49 2.850.0004 High Risk 4.98 2.91 0.0002 Intermediate Risk 6.24 2.95 0.0053

In these exisulind trials and several others involving the drug in otherindications, safety was evaluated by monitoring adverse events (AEs),clinical laboratory tests (hematology, serum chemistry, and urinalysis),vital signs (blood pressure, pulse rate, respiratory rate, temperature,and weight), physical examination, and upper endoscopy.

No outstanding safety issues have been demonstrated in the clinicaltrials conducted with exisulind to date in over 400 Patients. Exisulinddid not demonstrate any blood dyscrasia, dose-limiting vomiting, orneurological or renal toxicities associated with conventionchemotherapeutics. It also did not cause any clinically significantchanges in vital signs. In fact, in paired biopsies of polyp and normalcolonic tissues in APC patients, it was found that exisulind increasedapoptosis rates in polyp, but not normal colonic tissues, suggestingminimal effects on normal tissues.

At doses above the maximum tolerated dose (MTD=600 mg in patients withsubtotal colectomy; 400 mg in patients with intact colons; 350 mg inpediatric patients), the only dose-limiting adverse events found wereelevations in liver function tests (LFTs) that are seen early duringtreatment. When experienced, LFT elevations were rapidly reversible, anddo not recur when the dose has been lowered. Other events (e.g.,occasional abdominal pain) were typically short lasting and of mild tomoderate intensity, and did not necessitate discontinuing or lowering ofthe exisulind dose.

In short, these trials demonstrated that exisulind is an effective,well-tolerated chronic therapy for the clinical management of neoplasia.Thus, these results illustrate that selecting an additional compoundthat inter alia inhibits cGMP-specific PDE activity (as well as meetingthe other selection criteria of this invention) can result in atherapeutically effective drug, in vivo.

A second drug that was also invented before its mechanism of action wasfound to involve cGMP inhibition and before it was known to meet theselection criterion of this invention is(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride (Compound I). It has been demonstrated in in vitro and invivo evaluations as anti-neoplastic having activities against a broadrange of neoplasias. It is also safe in animal studies and in a single,escalating dose human study.

As one skilled in the art will recognize from the data presented below,Compound I can safely be given to animals at doses far beyond thetolerable (and in many cases toxic) doses of conventionalchemotherapeutics or anti-neoplastic NSAIDs. For example, in an acutetoxicity study in rats, single oral doses of Compound I administered (ina 0.5% carboxy-methylcellulose vehicle) at doses up to and including2000 mg/kg resulted in no observable signs of toxicity. At 4000 mg/kg,body weight gains were slightly reduced. A single dose of 1000 mg/kgadministered intraperitoneally resulted in reduced body weight gain,with mesenteric adhesions seen in some animals from this group atnecropsy.

In dogs, the administration of Compound I in capsules at 1000 mg/kgresulted in no signs of toxicity to the single group of two male and twofemale dogs. Due to the nature of Compound I capsules, this dosenecessitated the use of at least 13 capsules to each animal, which wasjudged to be the maximum number without subjecting the animals tostress. Therefore, these dogs were subsequently administered sevenconsecutive doses of 1000 mg/kg/day. At no time in either dosing phasewere any obvious signs of drug-related effects observed.

Thus, on a single-dose basis, Compound I is not acutely toxic. Based onthe findings of these studies, the oral LD₅₀ of Compound I wasconsidered to be greater than 1000 mg/kg in dogs and 4000 mg/kg in rats,and the intraperitoneal LD₅₀ was considered to be greater than 1000mg/kg in rats.

A seven-day dose-range finding study in rats, where Compound I wasevaluated by administering it at doses of 0, 50, 500 or 2000 mg/kg/dayresulting in no observable signs of toxicity at 50 mg/kg/day. At 500mg/kg/day, treatment-related effects were limited to an increase inabsolute and relative liver weights in female rats. At 2000 mg/kg/day,effects included labored breathing and/or abnormal respiratory sounds,decreased weights gains and food consumption in male rats, and increasedliver weights in female rats. No hematological or blood chemistrychanges nor any microscopic pathology changes, were seen at any doselevel.

A 28-day study in rats was also carried out at 0, 50, 500 and 2000mg/kg/day. There were no abnormal clinical observations attributed toCP-461, and body weight changes, ophthalmoscopic examinations,hematological and blood chemistry values and urinalysis examinationswere unremarkable. No macroscopic tissue changes were seen at necropsy.Organ weight data revealed statistically significant increase in liverweights at 2000 mg/kg/day, and statistically significant increases inthyroid weights for the 2000 mg/kg/day group. The slight increases atthe lower doses were not statistically significant. Histopathologicalevaluation of tissues indicated the presence of traces of follicularcell hypertrophy, increased numbers of mitotic figures (suggestive ofpossible cell proliferation) in the thyroid gland and mild centrilobularhypertrophy in the liver. These changes were generally limited to asmall number of animals at the 2000 mg/kg/day dose, although one femaleat 500 mg/kg/day had increased mitotic figures in the thyroid gland. Thefindings in the liver may be indicative of a very mild stimulation ofmicrosomal enzymes, resulting in increased metabolism of thyroidhormones, which in turn resulted in thyroid stimulation. Thus, oneskilled in the art will recognize that these effects are extremelyminimal compared to what one would expect at similar doses ofconventional chemotherapeutics or NSAIDs.

To further establish the safety profile of Compound I, a study wasperformed to evaluate whether Compound I-induced apoptosis of prostatetumor cell lines was comparable to its effects on prostate epithelialcells derived from normal tissue. The androgen-sensitive prostate tumorcell line, LNCaP (from ATCC (Rockville, Md.)) was propogated understandard conditions using RPMI 160 medium containing 5% fetal calveserum and 2 mM glutamine. Primary prostate epithelial cell cultures(PrEC) derived from normal prostate (from Clonetics Inc. (San Diego,Calif.)) were grown under the same conditions as the tumor cell lineexcept a serum-free medium optimized for the growth of such cultures wasused (Clonetics Inc). For the experiments, LNCaP or PrEC cells wereseeded in 96 well plates at a density of 10,000 cells per well. After 24hours, the cells were treated with either vehicle (0.1% DMSO) or 50 μMCompound I (free base) solubilized in DMSO. After various drug treatmenttimes (4, 24, 48, 72, or 99 hours) the cells were lysed and processedfor measurement of histone-associated DNA as an indicator of apoptoticcell death (see, Piazza et al., Cancer Research 57: 2452-2459, 1997).

FIG. 27 shows a time-dependent increase in the amount ofhistone-associated fragmented DNA in LNCaP cell cultures followingtreatment with 50 μM Compound I(free base). A significant increase infragmented DNA was detected after 24 hours of treatment, and theinduction was sustained for up to 4 days of continuous treatment. Bycontrast, treatment of PrEC (“normal″” prostate) cells with Compound I(50 μM) did not affect DNA fragmentation for up to 4 days of treatment.These results demonstrate a selective induction of apoptosis inneoplastic cells, as opposed to normal cells. This is in marked contrastto conventional chemotherapeutics that induce apoptosis or necrosis inrapidly growing normal and neoplastic cells alike.

Finally as to safety, in a single, escalating dose human clinical trial,patients, human safety study in which the drug was taken orally,Compound I produced no significant side effects at any dose, includingdoses above the level predicted to be necessary to produce anti-cancereffects.

As indicated above, Compound I also exhibits potent anti-neoplasticproperties. The growth inhibition IC₅₀ value obtained for Compound I was0.7 μM in the SW-480 cell line. This result has been confirmed byevaluating Compound I in rodents using aberrant crypt foci (“ACF”) as anindicator of carcinogenesis (see, Bird, Cancer Lett. 37: 147-151, 1987).This established rodent model of azoxymethane (“AOM”)-inducedcarcinogenesis was used to assess the effects of Compound I (free baseand salt) on colon cancer development in vivo. ACF are precursors tocolonic tumors, and ACF inhibition is predictive of chemo-preventiveefficacy.

In the rats in this experiment, ACF initiation was achieved by twoconsecutive weekly injections of the carcinogen. Compound I wasadministered one week prior to ACF initiation and for the duration ofthe experiment. ACFs were scored after 5 weeks of treatment. Compound Iwas administered orally to male Fisher 344 rats in the rat chow. Dailyfood consumption (mg/kg body weight) varied over the course of thestudy, and therefore Compound I dose was expressed a grams per kg ofdiet to provide a basis of comparison between doses. To determine ifCompound I had an adverse effect on growth and/or feeding behavior, bodyweight was determined throughout the course of the experiment. Theexperimental groups gained less weight than the controls, which wasindicative of bioavailability. However, the weight differences were lessthan 10% and not considered to affect ACF formation.

The free base of Compound I inhibited ACF formation as measured by areduction of crypts per colon. The data are summarized in Table 11. Withthe exception of the low dose group (only 0.5 g/kg diet), thedifferences between treatment and control groups were substantial, andstatistically significant in the case of the 1.0 and 2.0 g/kg dietgroup.

TABLE 11 Inhibition of Aberrant Crypt Foci by Compound I Compound MeanACF/colon p (t-test) Dose (g/kg diet) n (+SE) % Control vs. controlControl 10 149 ± 9  — — 0.5 7 149 ± 14 100 0.992 1 10 111 ± 9  75 0.0081.5 10 132 ± 4  89 0.101 2.0 10 107 ± 15 72 0.029

Also, Compound I retrospectively met the selection criterion of thisinvention, and was one of the compounds used to establish the validityof this selection criteria. For example, using the protocols describedpreviously, Compound I has a cGMP-specific PDE IC₅₀ value of 0.68 μMutilizing cGMP-specific PDE from HT29 cell extracts. Its COX Iinhibition (at 100 μM) was less than 25%.

As for being pro-apoptotic, Compound I's DNA fragmentation EC₅₀ was 15μM. In addition, the percent apoptosis for Compound I in SW-480 is shownin Table 12 at various drug concentrations.

TABLE 12 Apoptosis Induction of HT-29 Cells of SW-480 ColonAdenocarcinoma Cells by Compound I as Determined by Morphology TreatmentDose % Apoptosis Vehicle (0.1% DMSO) — 1 Compound I 0.35 μM  16 CompoundI 0.7 μM 27 Compound I 1.5 μM 88

Compound I's activity is not confined to activity against colon cancercell lines or animal models of colon cancer. It has a broad range ofanti-neoplastic effects in various neoplastic cell lines. Various typesof human cancer cell lines were propagated under sterile conditions inRPMI 1640 medium with 10% fetal bovine serum, 2 mM L-glutamine andsodium bicarbonate. To determine growth inhibitory effects of CompoundI, cells were seeded in 96-well plates at a density of 1000 cells perwell. Twenty-four hours after plating, the cells were dosed with variousconcentrations of the free base of Compound I solubilized in DMSO (finalconcentration 0.1%). The effect of the drug on tumor cell growth wasdetermined using the neutral red cytotoxicity assay following five daysof continuous treatment. Neutral red is a dye that is selectively takenup by viable cells by an ATP-dependent transport mechanism.

As summarized in Table 13, Compound I (free base) displayed potentgrowth inhibitory activity when evaluated against a panel of culturedhuman cell lines derived from various tissue origins. Compound Idisplayed comparable growth inhibitory effects regardless of thehistogenesis of the tumor from which the cell lines were derived. TheGI₅₀ value (concentration of drug to inhibit growth by 50% relative tovehicle control) calculated for all cell lines was 1-2 μM.

In addition to the data in the table below, we observed comparablesensitivity of human leukemia cell lines (CCRF-CEM, K562, and Molt-4), amyeloma cell line (RPMI8226), a pancreatic tumor cell line (PAN-1), andan ovarian tumor cell line (OVCAR-3) to Compound I (HCl salt).

TABLE 13 Growth Inhibition of Various Human Tumor Cell Lines by CompoundI Cell Line Tumor origin GI₅₀μM GI₉₀μM Colo 205 Colon 1.6 2.4 HCT-15Colon 1.7 3.0 HT-29 Colon 2.1 8.0 SW-620 Colon 1.7 2.5 DU145 Prostate1.6 2.8 PC-3 Prostate 1.7 82.5 NCI-H23 Lung 1.7 2.5 NCI-H322M Lung 2.113.2 NCI-H460 Lung 1.9 30.0 NCI-H82 Lung 1.7 5.8 MDA-MB-231 Breast 1.877.6 MDA-MB-435 Breast 1.6 2.3 UISO-BCA-1 Breast 1.5 4.7 Molt-4*Leukemia 1.6 ND CCRF-CEM* Leukemia 1.4 ND K-562* Leukemia 1.8 NDRPMI-8226* Myeloma 1.2 ND OVCAR* Ovary 1.2 ND PANC-1* Pancreas 2.2 ND*Testing was done with the free base of the compound unless otherwiseindicated with an asterisk in which case testing was done with the HClsalt.

Given the animal and human safety characteristics, and the animal andvery broad cell culture efficacy of Compound I, it is clear thatcompounds meeting the selection criteria of this invention (includingcGMP-specific PDE inhibition) can are useful anti-neoplastictherapeutics.

As to identifying structurally additional cGMP-specific PDE inhibitingcompounds that can be effective therapeutically as anti-neoplastics, oneskilled in the art has a number of useful model compounds disclosedherein (as well as their analogs incorporated by reference) that can beused as the bases for computer modeling of additional compounds havingthe same conformations but different chemically. For example, softwaresuch as that sold by Molecular Simulations Inc. release of WebLab®ViewerPro™ includes molecular visualization and chemical communicationcapabilities. Such software includes functionality, including 3Dvisualization of known active compounds to validate sketched or importedchemical structures for accuracy. In addition, the software allowsstructures to be superimposed based on user-defined features, and theuser can measure distances, angles, or dihedrals.

In this situation, since the structures of other active compounds aredisclosed above, one can apply cluster analysis and 2D and 3D similaritysearch techniques with such software to identify potential newadditional compounds that can then be screened and selected according tothe selection criteria of this invention. These software methods relyupon the principle that compounds, which look alike or have similarproperties, are more likely to have similar activity, which can beconfirmed using the selection criterion of this invention.

Likewise, when such additional compounds are computer modeled, many suchcompounds and variants thereof can be synthesized using knowncombinatorial chemistry techniques that are commonly used by those ofordinary skill in the pharmaceutical industry. Examples of a fewfor-hire combinatorial chemistry services include those offered by NewChemical Entities, Inc. of Bothell Washington, Protogene Laboratories,inc., of Palo Alto, Calif., Axys, Inc. of South San Francisco, Calif.,Nanosyn, Inc. of Tucson, Ariz., Trega, Inc. of San Diego, Calif., andRBI, Inc. of Natick, Mass. There are a number of other for-hirecompanies. A number of large pharmaceutical companies have similar, ifnot superior, in-house capabilities. In short, one skilled in the artcan readily produce many compounds for screening from which to selectpromising compounds for treatment of neoplasia having the attributes ofcompounds disclosed herein. To further assist in identifying compoundsthat can be screened and then selected using the criterion of thisinvention, knowing the binding of selected anti-neoplastic compounds toPDE5 Protein is of interest. By the procedures discussed below, it wasfound that preferable, desirable compounds meeting the selectioncriteria of this invention bind to the cGMP catalytic region of PDE5.

To establish this, a PDE5 sequence that does not include the catalyticdomain was used. One way to produce such a sequence is to express thatsequence as a fision protein, preferably with glutiathione S-transferase(“GST”), for reasons that will become apparent.

RT-PCR method is used to obtain the cGB domain of PDE5 with forward andreverse primers designed from bovine PDE5 A cDNA sequence(McAllister-Lucas L. M. et al, J. Biol. Chem. 268, 22863-22873, 1993)and the selection among PDE 1-10 families. 5′-3′, Inc. kits for totalRNA followed by oligo (dT) column purification of mRNA are used withHT-29 cells. Forward primer (GAA-TTC-TGT-TAG-AAA-AGC-CAC-CAG-AGA-AAT-G,203-227) and reverse primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC-CTC-TGC-TG,1664-1686) are used to synthesize the 1484 bp fragment coding for thephosphorylation site and both low and high affinity cGMP binding sitesof human PDE5 A (203-1686 bp, cGB-PDE5). The synthesized cGB-PDE5nucleotide fragment codes for 494 amino acids with 97% similarity tobovine PDE5 A. It is then cloned into pGEX-5X-3glutathione-S-transferase (GST) fusion vector (Pharmacia Biotech )withtac promoter, and EcoRI and XhoI cut sites. The fusion vector is thentransfected into E. Coli BL21 (DE3) bacteria (Invitrogen). Thetransfected BL21 bacteria is grown to log phase, and then IPTG is addedas an inducer. The induction is carried at 20° C. for 24 hrs. Thebacteria are harvested and lysed. The soluble cell lysate is incubatedwith GSH conjugated Sepharose 4 B (GSHSe-pharose 4B). The GST-cGB-PDE5fusion protein can bind to the GSH-Sepharose beads, and the otherproteins are washed off from beads with excessive cold PBS.

The expressed GST-cGB-PDE5 fusion protein is displayed on 7.5% SDS-PAGEgel as an 85 Kd protein. It is characterized by its cGMP binding andphosphorylation by protein kinases G and A. It displays two cGMP bindingsites, and the K_(d) is 1.6±0.2 μM, which is close to K_(d)=1.3 μM ofthe native bovine PDE5. The GST-cGB-PDE5 on GSH-conjugated sepharosebeads can be phosphorylated in vitro by cGMP-dependent protein kinaseand cAMP-dependent protein kinase A. The K_(m) of GST-cGB-PDE5Phosphorylation by PKG is 2.7 μM and Vmax is 2.8 μM, while the k_(m) ofBPDEtide phosphorylation is 68 μM. The phosphorylation by PKG showsmolecular phosphate incorporated into GST-cGB-PDE5 Protein on aone-to-one ratio.

A cGMP binding assay for compounds of interest (Francis S. H. et al, J.Biol. Chem. 255, 620-626, 1980) is done in a total volume of 100 μLcontaining 5 mM sodium phosphate buffer (pH=6.8), 1 mM EDTA, 0.25 mg/mLBSA, H³-cGMP (2 μM, NEN) and the GST-cGB-PDE5 fusion protein (30μg/assay). Each compound to be tested is added at the same time as³H-cGMP substrate, and the mixture is incubated at 22° C. for 1 hour.Then, the mixture is transferred to Brandel MB-24 cell harvester withGF/B as the filter membrane followed by 2 washes with 10 μL of cold 5 mMpotassium buffer(pH 6.8). The membranes are then cut out and transferredto scintillation vials followed by the addition of 1 mL of H₂O and 6 mLof Ready Safe™ liquid scintillation cocktail to each vial. The vials arecounted on a Beckman LS 6500 scintillation counter.

For calculation, blank samples are prepared by boiling the bindingprotein for minutes, and the binding counts are <1% when compare tounboiled protein. The quenching by filter membrane or other debris arealso calibrated.

PDE5 inhibitors, sulfide, exisulind, Compound B, Compound E, E4021 andzaprinast, and cyclic nucleotide analogs, cAMP, cyclic IMP,8-bromo-cGMP, cyclic UMP, cyclic CMP, 8-bromo-cAMP, 2′-O-butyl-cGMP and2′-O-butyl-cAMP are selected to test whether they could competitivelybind to the cGMP binding sites of the GST-cGB-PDE5 Protein. The resultswere shown in FIG. 24. cGMP specifically binds GST-cGB-PDE5 Protein.Cyclic AMP, cUMP, cCMP, 8-bromo-cAMP, 2′-O-butyl-cAMP and2′-O-butyl-cGMP did not compete with cGMP in binding. Cyclic IMP and8-bromo-cGMP at high concentration (100 μM) can partially compete withcGMP (2 μM) binding. None of the PDE5 inhibitors showed any competitionwith cGMP in binding of GST-cGB-PDE5. Therefore, they do not bind to thecGMP binding sites of PDE5.

However, Compound E does competitively (with cGMP) bind to PDE 5 (i.e.,peak A). (Compound E also competitively (with cGMP) binds to PDE peakB.). Given that Compound E does not bind to the cGMP-binding site ofPDE5, this the fact that there is competitive binding between Compound Eand cGMP at all means that desirable compounds such as Compound E bindto the cGMP catalyic site on PDE5, information that is readilyobtainable by one skilled in the art (with conventional competitivebinding experiments) but which can assist one skilled in the art morereadily to model other compounds. Thus, with the chemical structures ofdesirable compounds presented herein and the cGMP binding siteinformation, one skilled in the art can model, identify and select(using the selection criteria of this invention) other chemicalcompounds for use as therapeutics.

Compounds selected in accordance with the methodology of this inventionmay be formulated into pharmaceutical compositions as is well understoodfrom the ordinary meaning of the term “pharmaceutical composition” i.e.,a compound (e.g., like the solids described above) and apharmaceutically acceptable carrier for delivery to a patient by oraladministration in solid or liquid form, by IV or IP administration inliquid form, by topical administration in ointment form, or by rectal ortopical administration in a suppository formulation. Carriers for oraladministration are most preferred.

As is well known in the art pharmaceutically acceptable carriers inpharmaceutical compositions for oral administration include capsules,tablets, pills, powders, troches and granules. In such solid dosageforms, the carrier can comprise at least one inert diluent such assucrose, lactose or starch. Such carriers can also comprise, as isnormal practice, additional substances other than diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, troches and pills, the carriers may also comprise bufferingagents. Carriers such as tablets, pills and granules can be preparedwith enteric coatings on the surfaces of the tablets, pills or granules.Alternatively, the enterically-coated compound can be pressed into atablet, pill, or granule, and the tablet, pill or granules foradministration to the patient. Preferred enteric coatings include thosethat dissolve or disintegrate at colonic pH such as shellac or EudragetS.

Pharmaceutically acceptable carriers in pharmaceutical compositionsinclude liquid dosage forms for oral administration, e.g.,pharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs containing inert diluents commonly used in the art, such aswater. Besides such inert diluents, compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Pharmaceutically acceptable carriers in pharmaceutical compositions forIV or IP administration include common pharmaceutical saline solutions.

Pharmaceutically acceptable carriers in pharmaceutical compositions fortopical administration include DMSO, alcohol or propylene glycol and thelike that can be employed with patches or other liquid-retainingmaterial to hold the medicament in place on the skin so that themedicament will not dry out.

Pharmaceutically acceptable carriers in pharmaceutical compositions forrectal administration are preferably suppositories that may contain, inaddition to the compounds of this invention excipients such as cocoabutter or a suppository wax, or gel.

A pharmaceutically acceptable carrier and compounds of this inventionare formulated into pharmaceutical compositions in unit dosage forms foradministration to a patient. The dosage levels of active ingredient(i.e., compounds selected in accordance with this invention) in the unitdosage may be varied so as to obtain an amount of active ingredienteffective to achieve neoplasia-eliminating activity in accordance withthe desired method of administration (i e., oral or rectal). Theselected dosage level therefore depends upon the nature of the activecompound administered (e.g., its IC₅₀, which can be readilyascertained), the route of administration, the desired duration oftreatment, and other factors. If desired, the unit dosage may be suchthat the daily requirement for active compound is in one dose, ordivided among multiple doses for administration, e.g., two to four timesper day. For IV administration, an initial dose for administration canbe ascertained by basing it on the dose that achieves the IC₅₀ in theplasma contents of the average adult male (i.e., about 4 liters).Initial doses of active compound selected in accordance with thisinvention can range from 0.5-600 mg.

The pharmaceutical compositions of this invention are preferablypackaged in a container (e.g., a box or bottle, or both) with suitableprinted material (e.g., a package insert) containing indications,directions for use, etc.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

We claim:
 1. A method of treating neoplasia in a patient, comprisingadministering to the patient a pharmacologically effective amount of acompound selected by: determining COX inhibitory activity of thecompound, and determining PDE-5 inhibitory activity of said compound,wherein the compound that is selected for administration to the patientinhibits COX activity no more than 25% at a concentration of 100 μM andinhibits PDE-5 activity to a greater extent than exisulind, with theproviso that the compound is not sulindac or sulindac sulfide.
 2. Amethod for treating neoplasia in a patient, comprising administering tothe patient a pharmcologically effective amount of a compound selectedby determining the neoplastic growth inhibitory activity of thecompound; determining the PDE-5 inhibition activity of the compound; andselecting the compound for treating neoplasia that exhibits growthinhibitory activity and PDE-5 inhibitory activity, with the proviso thatsaid compound is not sulindac, sulindac sulfide or exisulind.
 3. Amethod for treating neoplasia in a patient, comprising administering tothe patient a pharmcologically effective amount of a compound selectedby determining the PDE5 inhibitory activity and the antineoplasticactivity of said compound and selecting the compound with PDE5inhibitory and antineoplastic activities, with the proviso that saidcompound is not sulindac, sulindac sulfide or exisulind.
 4. A method fortreating neoplasia in a patient, comprising administering to the patienta pharmcologically effective amount of a compound selected by: selectinga compound with PDE5 inhibiting activity; evaluating the neoplastic cellgrowth inhibiting activity of the compound; and selecting the PDE5inhibiting compound that inhibits neoplastic cell growth with theproviso that said compound is not sulindac, sulindac sulfide orexisulind.
 5. A method for treating neoplasia in a patient, wherein saidneoplasia is selected from the group consisting of colo-rectal, breastand prostate neoplasia, comprising administering to a patient apharmacologically effective amount of a compound selected by:determining COX inhibitory activity of the compound, and determiningPDE-5 inhibitory activity of said compound, wherein the compound that isselected for administration to the patient inhibits COX activity no morethan 25% at a concentration of 100 μM and inhibits PDE-5 activity to agreater extent than exisulind, with the proviso that the compound is notsulindac or sulindac sulfide.
 6. A method for treating neoplasiaselected from the group consisting of colo-rectal, breast and prostateneoplasia in a patient, comprising administering to the patient apharmcologically effective amount of a compound selected by determiningthe neoplastic growth inhibitory activity of the compound; determiningthe PDE-5 inhibition activity of the compound; and selecting thecompound for treating neoplasia that exhibits growth inhibitory activityand PDE-5 inhibitory activity, with the proviso that said compound isnot sulindac, sulindac sulfide or exisulind.
 7. A method for treatingneoplasia selected from the group consisting of colo-rectal, breast andprostate neoplasia in a patient, comprising administering to the patienta pharmcologically effective amount of a compound selected bydetermining the PDE5 inhibitory activity and the antineoplastic activityof said compound and selecting the compound with PDE5 inhibitory andantineoplastic activities, with the proviso that said compound is notsulindac, sulindac sulfide or exisulind.
 8. A method for treatingneoplasia selected from the group consisting of colo-rectal, breast andprostate neoplasia in a patient, comprising administering to the patienta pharmcologically effective amount of a compound selected by: selectinga compound with PDE5 inhibiting activity; evaluating the neoplastic cellgrowth inhibiting activity of the compound; and selecting the PDE5inhibiting compound that inhibits neoplastic cell growth, with theproviso that said compound is not sulindac, sulindac sulfide orexisulind.
 9. A method for treating colo-rectal neoplasia in a patientcomprising administering to the patient a pharmacologically effectiveamount of a compound selected by: determining COX inhibitory activity ofthe compound, and determining PDE-5 inhibitory activity of saidcompound, wherein the compound that is selected for administration tothe patient inhibits COX activity no more than 25% at a concentration of100 μM and inhibits PDE-5 activity to a greater extent than exisulind,with the proviso that the compound is not sulindac or sulindac sulfide.10. A method for treating colo-rectal neoplasia in a patient, comprisingadministering to the patient a pharmacologically effective amount of acompound selected by determining the neoplastic growth inhibitoryactivity of the compound; determining the PDE-5 inhibition activity ofthe compound; and selecting the compound for treating colo-rectalneoplasia that exhibits growth inhibitory activity and PDE-5 inhibitoryact ivity, with the proviso that said compound is not sulindac, sulindacsulfide or exisulind.
 11. A method for treating colo-rectal neoplasia ina patient, comprising administering to the patient a pharmacologicallyeffective amount of a compound selected by determining the PDE5inhibitory activity and the antineoplastic activity of said compound andselecting the compound with PDE5 inhibitory and antineoplasticactivities, with the proviso that said compound is not sulindac,sulindac sulfide or exisulind.
 12. A method for treating colo-rectalneoplasia, comprising administering to the patient a pharmacologicallyeffective amount of a compound selected by: selecting a compound withPDE5 inhibiting activity; evaluating the neoplastic cell growthinhibiting activity of the compound; and selecting the PDE5 inhibitingcompound that inhibits neoplastic cell growth, with the proviso thatsaid compound is not sulindac, sulindac sulfide or exisulind.